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Glutamate decarboxylase confers acid tolerance and enhances survival of

mycobacteria within macrophages

Rupal Rai, Bijina J. Mathew, Rashmi Chourasia, Anirudh K. Singh, Shivendra K.

Chaurasiya
PII: S0021-9258(25)00187-5
DOI: https://doi.org/10.1016/j.jbc.2025.108338
Reference: JBC 108338

To appear in: Journal of Biological Chemistry

Received Date: 12 November 2024

Revised Date: 3 February 2025

Please cite this article as: Rai R, Mathew BJ, Chourasia R, Singh AK, Chaurasiya SK, Glutamate
decarboxylase confers acid tolerance and enhances survival of mycobacteria within macrophages,
Journal of Biological Chemistry (2025), doi: https://doi.org/10.1016/j.jbc.2025.108338.

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┬® 2025 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and
Molecular Biology.
 Glutamate decarboxylase confers acid tolerance
and enhances survival of mycobacteria within
macrophages
Rupal Rai1, Bijina J. Mathew1, Rashmi Chourasia2, Anirudh K. Singh3, Shivendra K.
Chaurasiya1*
1
Molecular Signalling lab, Department of Biological Sciences and Engineering, Maulana
Azad National Institute of Technology Bhopal, M.P, India
2
Department of Basic Science, IES College of Technology, Bhopal, M.P, India
3
School of Sciences, SAM Global University, Raisen, M.P, India
*Corresponding author – [email protected]
Corresponding author address - Molecular Signalling lab, Department of
Biological Science and Engineering, Maulana Azad National Institute of

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Technology Bhopal, M.P, India.

Abstract

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Host-induced metabolic adaptations are crucial for Mycobacterium tuberculosis (Mtb) survival
and drug resistance. Mtb's persistence in the acidic environments of phagosomes and
phagolysosomes suggests its initial metabolic adjustments respond to acidic stress. Glutamate
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decarboxylase (Gad) enzyme, converts glutamate to GABA while consuming a proton, helping
regulate intracellular pH in bacteria. However, the role of Gad in mycobacteria has been
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unexplored. In this study, we investigated the function of Gad in Mtb and Mycobacterium
smegmatis (MS), which are encoded by Rv3432c (gadB) and MSMEG_1574 (gadA), an
orthologue of gadB, respectively. We observed upregulation of gad in both Mtb and MS under
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acidic stress and during infection within macrophages. Additionally, the expression of genes
involved in glutamate metabolism and the GABA shunt, such as glutamine synthetase
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(glnA1), glutamate dehydrogenase (gdh), glutamate synthase (gltD/B), GABA-

aminotransferase (gab-T), succinic semialdehyde dehydrogenase (gabD1/gabD2), α-
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ketoglutarate dehydrogenase (kdh), and 2-oxoglutarate dehydrogenase (sucA), were responsive

to acidic conditions, reflecting a metabolic shift. Similar gene expression patterns were
observed during macrophage infection. These findings suggest that Gad plays a role in
mycobacterial acid stress response. To further elucidate this, we generated an
MS gadA knockout strain (MSΔgadA) using allelic exchange. MSΔgadA exhibited reduced
survival at pH 3.0, a phenotype rescued by gene complementation. MSΔgadA also showed
decreased survival within macrophages. Additionally, Mycobacterium bovis BCG, which lacks
native Gad expression, demonstrated enhanced intracellular survival when overexpressing
Mtb gadB. These results suggest that Gad confers acid tolerance and promotes intracellular
survival in mycobacteria, highlighting its potential role in host adaptation.

Keywords - Mycobacterium tuberculosis, Mycobacterium smegmatis, tuberculosis,

phagocytosis, bacterial metabolism, bacterial pathogenesis, host‐pathogen interaction,
glutamate decarboxylase, acid resistance, acid tolerance

Running title
Rv3432c promotes mycobacterial survival in macrophages
 1. Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains the second leading
cause of death from a single infectious agent worldwide (1). The primary challenges hindering
the global eradication of this otherwise curable disease include the prolonged treatment
regimen, which spans 1 to 6 months leading to non-compliance, and the alarming rise in
multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR)
strains of TB (2). Thus, the world is facing an urgent need for new approaches and drugs to
combat TB, which in turn requires a greater understanding of the biochemical and
physiological processes that enable Mtb to survive the stressful environment within
macrophages and acquire drug tolerance/resistance (3–5). The interaction between Mtb and its
host/environment can result in multiple outcomes. It is of utmost importance to understand
each point of interaction with the host. In the pre-antibiotic era, Mtb faced natural selection
pressures, primarily the host's immune response, as it became internalized by macrophages.

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While in the post-antibiotic era, Mtb developed drug resistance, mainly by acquiring mutations

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in drug targets (6, 7). However, before exposure to therapeutic drugs, the pathogen must first
survive from the protective responses of macrophages (8, 9). Mycobacteria exhibit remarkable
ability to adapt and manipulate host cells, enabling their survival (10). It is believed that

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pathogenic Mtb strains have evolved from environmental saprophytic mycobacteria under
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selection pressure in an ever-changing environment (11). Even one of the most pathogenic
clades of Mtb - the Beijing clade - is known to arise due to physiological and molecular changes
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that occur as a result of selective pressure, leading to increased hypervirulence (7).
As soon as bacilli enter into the host, they are engulfed by macrophages where they get exposed
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to oxidative (12) and acidic stress (13). These components are integral to the immune defence,
with acidification signalling the onset of macrophage activity and coordinating with other
immune responses (14). The ability of Mtb to adapt and cope with these stresses is crucial for
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the establishment of infection and the progression of the disease (15). Mtb faces acidic stress
in phagosomes within macrophages stimulated with IFN-γ and/or at the centres of caseating
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granulomas. Being a part of the host defence, phagosome acidification is an important

environmental condition. Once bacilli are engulfed, the phagosome pH drops to 6.0 (16).
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Several known factors contribute to the acidification of phagosomes, including the vacuolar
proton-ATPase, the Na+/H+ exchanger, and proton-conductive pathways (17, 18).
Consequently, when phagosomes fuse with lysosomes, the intracellular pH decreases to
approximately 4.5 (19). Mtb is known to inhibit this fusion, thereby hindering phagosome
maturation and acidification. Nevertheless, various studies demonstrate that Mtb can survive
and replicate within the acidified environments of phagosomes and phagolysosomes, where the
pH ranges from 6.5 to 4.5 (20–22). Mtb experiences gradual acidification instead of a sudden
and lethal drop in pH within macrophages. It has been proposed that Mtb may develop induced
acid tolerance by adjusting its metabolism in response to acidic conditions (23, 24). However,
the underlying mechanisms remain poorly characterized and require further investigation in
Mtb. While acid tolerance and extreme acid resistance mechanisms are well-documented in
various other Gram-positive and Gram-negative bacteria (25–27), these are not yet fully
deciphered in Mtb. However, evidence of such adaptive acid tolerance mechanisms in Mtb
remains lacking, representing a critical area for future research in Mtb pathogenesis.
Protein kinase G (PknG) is a recognized virulence factor that enhances mycobacterial survival
within macrophages and contributes to antibiotic resistance (28–30). We recently established
that PknG regulates acid tolerance in mycobacteria (31). PknG was also shown to regulate
glutamate metabolism (32). Glutamate metabolism has also been linked with other stress
responses in Mtb (33, 34). The glutamate decarboxylase (Gad)-mediated conversion of
glutamate to GABA is a well-known acid resistance mechanism in bacteria (35, 36). While the
Mtb genome encodes gad, its role in mycobacteria is not well documented. Our previous work
demonstrated that Gad is expressed in both Mtb and Mycobacterium smegmatis (MS) (37). In
this study, we further demonstrated that Gad mediates acid tolerance in mycobacteria leading
to enhanced intracellular survival within macrophages.

2. Results

2.1 Gene expression analysis reveals increased gad expression and modulation of
glutamate metabolism-related genes in mycobacteria during acidic stress and
macrophage infection

The gene expression pattern under different conditions provides valuable insights into the
function of the gene. To investigate whether Gad is associated with the acidic stress response
in mycobacteria, we analyzed the expression of gad and other selected genes after exposure to

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acidic pH and during macrophage infection. These genes were chosen based on their

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established roles in glutamate metabolism and their potential involvement in acid stress
adaptation. The expression patterns of these genes may highlight interconnected pathways

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contributing to pH homeostasis and intracellular adaptation through Gad-mediated glutamate
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metabolism. Total RNA was isolated from mycobacterial cultures grown in acidic medium (pH
5.0 ±0.2) for 4 hours, whereas mycobacterial cultures grown in normal medium served as
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control for the gene expression analysis by qRT-PCR. It was observed that expression of gad,
GABA-aminotransferase (gab-T), glutamate dehydrogenase (gdh) and fatty acyl CoA
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synthetase (fadD9) are upregulated whereas expression of glutamate synthase (gltD), 2-

oxoglutarate dehydrogenase (sucA) are downregulated following exposure of MS to acidic
medium (Fig 1A). No significant change in expression of glutamine synthetase (glnA),
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glutamate synthase (gltD6458), succinic semialdehyde dehydrogenase (gabD2), isocitratelyase

(aceA) was observed in MS following exposure to acidic medium (Fig 1A). Upregulation of
gad, gaba-at along with other genes suggests that these changes may be a protective response
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in the stressed mycobacteria. Mycobacterial pathogens are also exposed to acidic stress during
infection in macrophages. To investigate if similar changes are exhibited by mycobacteria
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during infection of the macrophages, we analyzed the expression of these genes in MS during
infection of macrophages. Our results revealed comparable gene expression profile with
upregulation of gad, gaba-at, glnA, gabD2, fadD9, aceA, and downregulation of gdh, gltD,
gltD6458, sucA compared to control MS strain during macrophage infection (Fig 1B). Taken
together, these results indicate a possible role of gad in bacterial survival within the acidic
environment found in the macrophages.
We next sought to determine whether Mtb also exhibits a similar response during exposure to
acidic stress and infection of macrophages. Gene expression analysis of Mtb cells exposed to
acidic stress showed similar pattern of gene expression. The expression of gad, gab-T, gltD,
gltB, fadD9 and icl1 were upregulated whereas expression of glnA1, gdh, succinic
semialdehyde dehydrogenase (gabD1) were downregulated in Mtb cells following exposure to
acidic medium (Fig 1C). The expression of gabD2 and α-keto-glutarate dehydrogenase (kdh)
showed no changes in Mtb after exposure to acidic medium (Fig 1C). The upregulation of gad
following acidic stress suggests its role in Mtb’s response to acidic environment such as those
observed within macrophages. To further confirm gad expression in Mtb during infection, gene
expression analysis was performed in these bacteria during infection of macrophages, which
showed increased expression of gad, gdh, gltD, gltB and glnA1 (Fig. 1D).
Overall, the expression data of the selected genes clearly demonstrate alterations in glutamate
metabolism in mycobacteria during acidic stress and infection within host cells.
2.2 Ectopic overexpression of gad supports acidic tolerance in mycobacteria

The upregulation of gad in mycobacteria in the acidic medium suggests its role in acid tolerance
in mycobacteria. To further investigate whether the upregulation of gad during acidic stress
contributes to the ability of mycobacteria to survive under acidic environment, recombinant MS
overexpressing gadA or gadB were created. For this purpose, gadB from Mtb and gadA from
MS were cloned separately into an integrative vector, pMV361, followed by verification of the
recombinant clones by restriction enzyme digestion (S Fig 1). The pMV361 is a shuttle vector
used for mycobacterial studies enabling efficient expression of genes of interest under the
control of a constitutive hsp60 promoter. The recombinant plasmids were subsequently
electroporated into MS to yield kanR MS::gadA and MS::gadB strains. The presence of
recombinant pMV361:gadA and pMV361:gadB integrated in the respective strains, MS::gadA
and MS::gadB, was confirmed by colony PCR using specific primer pairs and genomic DNA

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as template. The results showed the presence of PCR amplicons with the DNA from only the

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recombinant strains whereas the empty vector control strain did not yield any PCR
amplification product (Fig 2A). Ectopic overexpression of gad in the recombinant strains was
then confirmed at the mRNA transcript level by qRT-PCR as well as at the protein level by the

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Gad assay. As presented in Fig. 2B, both MS::gadA and MS::gadB exhibited higher activity in
Gad assay compared to the MS::EV. Appearance of blue colour in Gad assay is an indicator of
positive Gad activity. In case of recombinant MS strains Gad reagent turned blue within 30
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minutes of incubation, compared to 4 h for MS::EV, thus confirming overexpression of Gad in
the recombinant strains (Fig 2B).
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Next, we examined the role of gad in acid tolerance. All three strains viz., MS::EV, MS::gadA
and MS::gadB were subjected to acid survival assay. Both MS::gadA and MS::gadB showed
significantly higher survival at the pH 3.0 when compared with MS:EV (p = 0.0229, p =
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0.0470). By CFU estimation it was found that the survival of Gad-overexpressing strains is
almost two-fold higher in comparison to the MS::EV (Fig 2C). These results thus indicate that
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Gad confers acid resistance in mycobacteria.

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2.3 Deletion of gad reduces survival of MS in acidic medium

To further confirm the role of gad in acid tolerance in mycobacteria, a gad deletion mutant of
MS (MSΔgadA) was constructed using the allelic exchange method. The schematic of allelic
exchange cassette for gadA deletion is shown in Fig. 3A. The deletion of gad in the knockout
strain was confirmed by Southern blotting (Fig. 3B) and Sanger sequencing. MSΔgadA was
complemented with either gadA gene (designated as MSΔgadA::gadA) or gadB
(MSΔgadA::gadB), which were cloned downstream of the hsp60 promoter in the pMV361
vector at an ectopic locus in the MSΔgadA genome. To confirm the loss of Gad activity in the
mutant and restoration in the MSΔgadA::gadA and MSΔgadA::gadB complemented strains,
these strains were subjected to the Gad assay. As expected, the Gad assay confirmed the loss
of Gad activity in MSΔgadA, which was restored by gad gene complementation in both
MSΔgadA::gadA and MSΔgadA::gadB (Fig. 3C). These observations confirmed the loss of the
gad gene and Gad activity in MSΔgadA. To test the contribution of Gad in supporting acid
resistance in mycobacteria, deletion mutant as well as the complemented strains were subjected
to acid survival assay. The mycobacterial cultures were grown until the OD600 reached 0.6-0.8,
and then sub cultured after thorough washing in the medium of either pH 7.0 ±0.2 or pH 3.0
±0.2 for 4 h. The viability of cultures was then determined by CFU plating on LBGT agar
plates. CFUs were enumerated after 3 days of incubation, and relative survival of the
mycobacterial strains was determined. Our results show lower survival of MSΔgadA at pH 3.0
±0.2 when compared to the wild-type MS (MS wt). A significant decrease in the survival of
MSΔgadA compared to MS wt at pH 3.0 (p = 0.0226), and the restoration of MSΔgadA
viability by complementation with gad gene from both MS and Mtb origin (p = 0.0057, p =
0.028 respectively) together establish the role of gad in acid tolerance in mycobacteria
(Fig.4A). Indeed, the complemented strains showed even more survival compared to MS wt
strain which may be due to the fact that complemented strain carries the gene under a strong
constitutively expressing promoter. These observations are in accordance with our previous
observations when we compared MS wt with gad overexpressing MS strains for their ability
to survive at acidic pH 3.0 (Fig. 2C). Previous studies suggest that pH 3.0 is lethal for
mycobacteria. To further analyse whether gad also supports mycobacterial growth at a
sublethal pH, mycobacterial cultures were exposed to pH 5.0 ±0.2 for 6 h. We observed
significantly reduced survival in MSΔgadA cultures treated with pH 5.0 when compared to MS
wt and complemented strain (p = 0.02, NS p = 0.3750 respectively) (Fig. 4B). These
observations confirm the role of gad in supporting mycobacterial adaptation and fitness to cope

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up with acidic environment.

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2.4 gad promotes intracellular survival of mycobacteria within macrophages

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During infection, Mtb is known to survive within macrophages. As soon as the pathogen enters
macrophages, the intracellular environment becomes acidic, which acts as a primary defence
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against the pathogen. Mtb is known to survive under the acidic environment within
phagosomes and phagolysosomes. Considering our earlier observations, we next sought to
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determine whether Gad activity can promote intracellular survival of mycobacteria within
macrophages. For this purpose, we infected THP-1 and J774.A.1 cells with recombinant MS
strains. We observed significantly lower survival of MSΔgadA in both THP-1 as well as
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J774.A.1 cells when compared to MS wt and gadA complemented counterparts. The survival
of MSΔgadA was almost two-fold less in J774.A.1 cells when compared to MS wt (p = 0.0417)
and this reduced survival was restored by gadA complementation (Fig. 5A). Similar pattern of
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survival was also observed in THP1 cells (p = 0.0045 respectively) (Fig. 5B). These findings
suggest that gad may promote intracellular survival of mycobacteria within macrophages.
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While MS is a useful model for studying mycobacteria, it does not naturally infect the host and
survive within macrophages for long. Hence, we further confirmed role of gad in intracellular
survival of BCG, which is an attenuated strain derived from the Mtb-complex bacterium M.
bovis, as a complementary model to support the findings obtained from the MS strain.
Importantly, BCG does not express Gad and tested negative Gad activity in the Gad assay. This
allowed us to ectopically express the gad gene and directly compare the intracellular survival
of recombinant BCG to wild-type BCG. Gad expressing BCG was constructed by
electroporating pMV361:gadB in BCG (BCG::gadB) and the integration of the gene was
confirmed by PCR (Fig. 5C). Gad activity in BCG::gadB as determined by the Gad assay
further confirmed the expression of gad in the recombinant BCG strain (Fig. 5D). The
BCG::gadB showed positive Gad activity while empty vector transfected BCG did not show
Gad activity (Fig 5D). Notably, expression of Gad increased the intracellular survival of BCG
in J774.A.1 cells by 1.8 fold (p = 0.0331) at 48 h post infection (Fig. 5E). These observations
corroborate our findings and further suggest that Gad promotes the intracellular survival of
mycobacteria within macrophages.
3 Discussion
Mtb has the ability to survive and multiply inside host macrophages for a long period of time.
Although drugs are available to treat TB infection with considerable success but the currently
used long treatment regime, high drug toxicity and emergence of drug resistance has alarmed
the need for new and better drugs (15). In order to discover new drugs and to reduce incidences
of drug resistance, new drug targets and mechanisms conferring drug resistance are needed to
be discovered. Metabolic pathways in bacterial pathogens have been implicated in adaptation
inside host cells and drug resistance (41). Pathogens alter their metabolic pathways to survive
host defence and to counter the effects of antibiotics (41, 42). The exposure of E. coli to low
pH promotes intrinsic resistance to antibiotics (44). Gad, a major component of the acid
resistance system in bacteria, has been associated with increased antibiotic tolerance in E. coli
(45). We earlier demonstrated that Gad is expressed in Mtb as well as in MS (37). The
expression of Gad in Mtb was detected at both acidic as well as at normal pH (37). In this study

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we aimed to investigate the expression of gad both in Mtb and MS, and its effect on bacterial

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survival under acid stress and during macrophage infection. Gene expression analysis revealed
that gad was upregulated in Mtb at acidic pH as well as within macrophages. Similarly, the
expression of gad in MS was also upregulated at acidic pH as well within macrophages. Gene

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expression analysis suggests the role of Gad in mycobacterial acid resistance as well as its role
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in altered metabolic tricarboxylic acid (TCA) cycle within mycobacteria.
Altered levels of metabolites of the TCA cycle have been shown to confer antibiotic resistance
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in Mtb (46). Mtb was proposed to lack Kdh activity limiting the conversion of α-ketoglutarate
to succinyl CoA (47). Instead, kdh codes for α -ketoglutarate decarboxylase which converts α-
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ketoglutarate into succinic semialdehyde (SSA) (47). Tian et al. proposed that α-ketoglutarate
decarboxylase bifurcates the TCA cycle by converting α-ketoglutarate into SSA (47).
Consistent with this finding, we observed downregulation of kdh/sucA in Mtb and MS at both
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acidic conditions and within macrophages. We also observed the upregulation of gab-T in Mtb
and MS during acidic stress and during infection within macrophages. gabD1/gabD2 was
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found to be upregulated during infection of MS within macrophages. The increased expression

of these genes suggests a coordinated metabolic response where Gad-mediated glutamate
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decarboxylation may be coupled with the TCA cycle via the GABA shunt. The increased
expression of gab-T and simultaneous decrease in kdh expression suggests that, under acidic
conditions, Mtb may metabolise α-ketoglutarate through Gab-T. Gab-T converts GABA and
α-ketoglutarate into SSA and glutamate. This process may provide an alternate pathway for the
TCA cycle which also regenerates glutamate for further decarboxylation by Gad. During acidic
stress, Mtb metabolizes glutamate to GABA via Gad, utilizing a proton, which may be
beneficial for the cell under acidic conditions by removing excess protons at low pH. It has
been inferred that GABA can neutralize protons and simultaneously contribute to the
NAD+/NADH balance, playing a role in Mtb’s early adaptation to both acidic and oxidative
stresses (48). Findings such as high levels of α-ketoglutarate also suggest the presence of an
alternate pathway, i.e., the GABA shunt, which has been identified as one of the metabolic
pathways for lactate and pyruvate metabolism in Mtb in studies combining microbial
physiology analysis with transposon-directed insertion site sequencing (49).
Mtb is known to alter or integrate its metabolic pathways depending on nutrient availability
and environmental stress (50). Changes in the expression of glutamate metabolism genes i,e
gdh, glnA/glnA1 and gltD/gltB in MS and Mtb during acidic stress and during infection suggest
similar metabolic adaptation in mycobacteria to the acidic environment encountered within
macrophages. Mycobacteria also face nutritional scarcity, and in order to survive, further
changes in metabolic pathways are reported (51, 52). Mtb cannot utilize lactate or pyruvate
under oxygen-limiting conditions. Transcriptomic and proteomic analyses reveal that Mtb
utilizes the glyoxylate shunt and methyl citrate cycle to metabolize lactate and pyruvate (49).
Mtb acquires glutamate from host macrophages, and high levels of glutamate was found in the
cell lysates of Mtb (47) which may serve as both a carbon and a nitrogen sources for its
intracellular survival within the host (53, 54). Glutamate can be used as both a carbon and
nitrogen source via GDH which converts glutamate to ammonium, thus providing protection
against acidic stress by buffering with ammonia. This also enables carbon assimilation by
degrading glutamate to 2-oxoglutarate (55). The depletion of intracellular glutamate in the
ΔgltBD double mutant and its accumulation in Δgdh mutant suggest that glutamate homeostasis
is critical under acidic stress conditions in BCG. Gdh was reported to be essential for optimal
growth in BCG and also contributes to nitrosative stress resistance and intracellular survival in
macrophages (55, 56). GltB/D facilitates the conversion of glutamine to glutamate, ensuring a
stable intracellular glutamate pool. Its role in GABA synthesis becomes critical under
propionate stress induced by the methylcitrate cycle (MCC) in BCG (34). The growth defects
caused by propionate toxicity were alleviated by monosodium glutamate supplementation,
highlighting the metabolic link between MCC and glutamate pathways. The conservation of

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glutamate pool may be utilised either by Gdh associated anaplerosis of TCA cycle

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intermediates or by Gad for proton neutralization (34). These mechanisms likely sustain TCA
activity and pH balance under acidic or nutrient-limited conditions. GlnA1 one of the four

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glutamine synthetase present in Mtb, is the only enzyme detected, isolated, and assayed in
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growing mycobacteria (57). In MS, glnA1 expression and glutamine synthetase activity are
tightly regulated by nitrogen availability, downregulated under excess ammonium and
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upregulated under nitrogen starvation (58). In Mtb, glnA1 has been associated with virulence
and intracellular survival. Mutants lacking glnA1 were attenuated in THP-1 macrophages and
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failed to infect guinea pigs (59). Moreover, glnA1 was shown to be essential for in vitro growth
and involved cell wall remodelling through poly-L-glutamate synthesis (57, 60). Proteomic
studies also indicate upregulation of glnA1 during acidic stress which are consistent with our
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observations of increased glnA1 expression in acidic conditions, suggesting its role in pH

adaptation and intracellular persistence (61).We observed the upregulation of fadD9 and icl1
in Mtb and MS during acidic stress. Deletion of icl1/2 impairs growth at acidic pH but not at
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neutral pH, highlighting the importance of anaplerotic metabolism during acidic stress (62).
While icl1/2 does not directly channel GABA into the TCA cycle, its upregulation, along with
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gabT, supports the hypothesis that alternate metabolic pathways are active under these
conditions to sustain TCA cycle function. Consistent with our observations, proteomic studies
by Choudhary et al. have also demonstrated the upregulation of FadD9 during acidic stress in
Mtb (61). FadD9 may channel fatty acid-derived acetyl-CoA into the TCA cycle, providing
carbon input and supporting energy metabolism under stress conditions. Icl and Gad thus may
also be linked with fatty acid metabolism as well, which suggests the possibility that fatty acids
may also be utilized as energy source during acidic stress in mycobacteria (49).
In the Mtb genome, the gene for Gab-T– the enzyme that catalyzes the first reaction of the
GABA shunt is located adjacent to the gene coding for FadD9 (the enzyme that catalyzes the
first step of the pathway, producing acetyl coA from fatty acids). This suggests the possibility
that both enzymes may be required for a common pathway. It is also noteworthy to mention
that Mtb genome does not code for a GABA/glutamate antiporter which is required to exchange
external glutamate for intracellular GABA and thereby reducing intracellular protons. This
suggests that GABA produced by the action of Gad must be utilized by other pathways. One
such probable pathway is the GABA shunt, which allows GABA to be consumed by the TCA
cycle. This pathway requires GABA-AT which catalyzes transfer of an amino group from
GABA to α-ketoglutarate to convert it into SSA and simultaneous regeneration of glutamate.
SSA is then converted to succinate and enters the TCA cycle. In this way, glutamate can be
continuously decarboxylated and regenerated leading to the consumption of protons and
increase in intracellular pH. This hypothetical model explains for absence of GABA/glutamate
anti-porter in Mtb (Fig. 6). The existence of such mechanisms has been shown in other bacteria
but not in mycobacteria (63–65).
The ability of Mtb to survive within the acidic and oxidative environment of host macrophages
is critical for its pathogenesis and the establishment of TB. One key adaptation mechanism that
has emerged from our work is the role of Gad in mediating acid resistance. Our results
demonstrate that the overexpression of Gad in MS confers a significant survival advantage in
acidic environments (pH 3.0). Similar, results were observed when Mtb Gad was expressed in
MS, suggesting a conserved function of Gad in mycobacteria. Conversely, a gad deletion
mutant of MS exhibited markedly reduced survival under acidic conditions, with the phenotype
being rescued upon complementation with the gad gene from either MS or Mtb origin. Our
results showed that Gad supports mycobacterial growth both at lethal (pH 3.0) and sub-lethal
(pH 5.0) pH. These findings underscore the role of Gad in acid resistance and tolerance,
indicating that this enzyme may be required for the survival of mycobacteria in acidic
environments. These observations suggest that Gad may also support the intracellular survival

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of mycobacteria within the host. The gad deletion in MS reduced the survival within

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macrophages, which was restored by gene complementation. Interestingly, our data show that
BCG, which lacks inherent Gad activity, exhibits enhanced survival within macrophages when

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Gad is ectopically overexpressed. This suggests that the Gad-mediated acid resistance
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mechanism not only enables mycobacteria to withstand the acidic pH of the phagosome but
may also contribute to the pathogen’s virulence by enhancing its ability to persist within the
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host. Previous studies have shown that PknG regulates multiple processes such as antibiotic
sensitivity, glutamate metabolism, acid tolerance, and intracellular survival, it suggests a
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common link between these adaptive responses in mycobacteria (30–32, 66–68).

Notably, gad expression has been shown to be increased in antibiotic-resistant E. coli,
indicating a potential role of this gene in resistance mechanisms (45). Furthermore, altered
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levels of GABA and TCA cycle intermediates, have been associated with antibiotic sensitivity
in mycobacteria (42, 69–71) thus raising the possibility that Gad, through its impact on
glutamate metabolism and intracellular pH regulation, might modulate metabolic states that
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influence mycobacterial responses to antibiotics. Further investigations will be needed to

understand if Gad contributes to antibiotic resistance in mycobacteria.
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4 Conclusion
Our study highlights the important role of Gad in mycobacterial survival under acidic stress
prevailing within host macrophages. By facilitating glutamate decarboxylation and maintaining
intracellular pH homeostasis, Gad enables mycobacteria to survive in hostile environments.
The previously reported upregulation of gad in antibiotic-resistant E. coli and the link between
altered GABA/TCA cycle intermediates and antibiotic sensitivity in mycobacteria suggest that
Gad may also contribute to antibiotic resistance. Further studies are required to elucidate the
role of Gad in the antibiotic response in mycobacteria. These findings underscore the
importance of Gad in mycobacterial physiology and suggest it as a potential target for
developing novel therapeutic strategies to combat TB, particularly the drug-resistant cases.

5 Materials and methods

5.1 Bacterial strains, culture media, and cell culture

MS strains were cultured in Luria-Bertani (LB) broth supplemented with 0.5% (v/v) glycerol
and 0.05% (v/v) tween 80. Mtb H37Rv and BCG strain were cultured in Middlebrooks 7H9
broth (Himedia) supplemented with 10% albumin-dextrose-catalase (ADC) (Himedia), 0.5%
glycerol (Himedia) and 0.05% Tween-80. Mtb H37Rv MC26206 (38) was cultured in
Middlebrook 7H9 supplemented with 1× oleic acid-albumin-dextrose-saline (OADS), 50
µg/ml leucine, 24 µg/ml pantothenate, 0.5% glycerol and 0.05% tyloxapol. Escherichia coli
(E. coli) DH5α cells was obtained from Himedia. All cultures were routinely tested for
contamination. E. coli strains were cultured in LB broth, pH 7.0 ±0.2 (Himedia). All cultures
were grown at 37°C with or without shaking at 220 rpm. Whenever required the pH of media
was adjusted to 5.0 ±0.2 and 3.0 ±0.2 using either HCl or 20 mM sodium citrate buffer before
autoclaving. Whenever needed, LB or LBGT (LB with 0.5% glycerol and 0.05% tween 80)
medium was supplemented with 1.5% agar (Himedia) in order to make semi-solid media for
culturing respective bacteria. Whenever needed 7H10 media was used supplemented with 10%
ADC, 0.5% glycerol and 0.05% tween-80. 25 µg/ml and 50 µg/ml kanamycin (kan), 100 µg/ml
ampicillin (amp), 50 µg/ml hygromycin (hyg), and 200 µg/ml amikacin were used, wherever
needed. All the restriction enzymes were obtained from NEB. All reagents used in the study
were procured from Sigma Aldrich unless mentioned otherwise. The primers used in the study

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are listed in supplementary table 1. All the plasmids and bacterial strains used in the study are

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listed in supplementary table 2.

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The THP-1 human macrophage-like cell line (ATCC strain acquired from the National Centre
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of Cell Science (NCCS), Pune) were cultured in RPMI-1640 medium (Gibco, USA)
additionally adding 2.0 g/L sodium bicarbonate supplemented with 10% heat inactivated fetal
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bovine serum (FBS) (Hi-FBS, Gibco) (complete RPMI) at 37°C, 5% CO2. THP-1 cells were
differentiated by treatment with 50 nM Phorbol 12- Myristate, 13-acetate (PMA) (Sigma, USA)
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for 48 hours (h) before using for the experiments. The J774.A.1 mouse derived macrophage
cell line (ATCC strain acquired from the NCCS, Pune) were cultured in DMEM medium
(Gibco, USA) additionally adding 3.7 g/L sodium bicarbonate supplemented with 10% heat
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inactivated FBS (complete DMEM) at 37°C, 5% CO2. All cell lines were routinely verified for
their characteristics and tested for contamination.
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5.2 Cloning of gad in MS and BCG strains

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The gadB gene was amplified from Mtb genomic DNA and gadA was amplified from MS
genomic DNA using phusion polymerase enzyme. PCR amplicon amplified using gene specific
oligonucleotides was cloned in pJET1.2/blunt end cloning vector using T4 DNA ligase enzyme
(NEB). Both DNA fragments of ~1.4 kb were obtained by EcoRI and HindIII digestion and
cloned at the same sites in pMV361 vector separately. E. coli DH5α was used for propagation
of the recombinant clones. The transformants were selected on LB agar supplemented with
50µg/ml kan after overnight incubation at 37°C. The integrity of clone was confirmed by
restriction enzyme digestion and Sanger sequencing. The confirmed pMV361:gadB,
pMV361:gadA plasmid and pMV361 empty vector (EV) were electroporated into MS MC2155
at 2500 volts using Eporator Electroporator (Eppendorf) and selected on LBGT agar plates
with 25 µg/ml kan. The clone pMV361:gadB and pMV361 (EV) were also electroporated in
BCG strain at 2500 volts and selected on 7H10 agar plates with 25 µg/ml kan. Confirmation
of recombinant was done by colony PCR using vector and gene specific primers.

5.3 Gad assay

The Gad assay was performed as described previously by Cotter et.al (35). The method was
optimized for mycobacterial cultures as described previously (37). Briefly, the test solution
containing 1 g of L-glutamic acid (MP Biomedicals), 0.3 ml of Triton X-100, 90 g of NaCl and
0.05 g of bromocresol green indicator (MP Biomedicals), in 1 l of water was prepared (Gad
reagent). Gad reagent was adjusted to pH 4.5 ±0.2 using NaOH. Mycobacterial cultures were
grown till OD600 reached 0.8-1.0. 1ml of cells were harvested by centrifugation at 6000 rpm
for 10 min (minutes) followed by washing with phosphate buffer saline (PBS). Cells were then
resuspended in 500µl Gad reagent followed by incubation at 37°C. The appearance of a blue
colour within 4 h of incubation was considered positive for Gad activity. The bromocresol
green dye serve as an indicator for change in pH of the solution. E. coli served as the positive
control, while assays without cells, without L-glutamate, or with D-glutamate functioned as
negative controls (D- glutamate is not a substrate but an inhibitor of Gad).
5.4 Generation of gadA deletion mutant in MS
An allelic exchange strategy was applied to generate gadA deletion mutant of MS. The 2407
bp sequence was amplified from MS genomic DNA containing 1380 bp of gadA with 502 bp
upstream of the gene and 525 bp downstream of the gene as flanking regions using phusion
polymerase enzyme (NEB). The PCR amplicon amplified using gene specific oligonucleotides
was cloned in pJET1.2/blunt end cloning vector using T4 DNA ligase enzyme (NEB). E.
coli DH5α was used for propagation of the recombinant clones. Transformants were selected

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in the presence of the amp on LB agar plates after overnight incubation at 37°C. Plasmid was
isolated and inverse PCR was performed using phusion polymerase enzyme with specific
oligonucleotides, deleting 1236 bp of gadA. The purified inverse PCR product was ligated with

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hyg cassette of 1026 bp using T4 DNA ligase and transformed in E. coli DH5α cells. Before
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ligation hyg cassette was amplified using specific oligonucleotides, digested by EcoRV and
purified. Transformants were selected in the presence of the amp and hyg on LB agar plates
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after overnight incubation at 37°C. Plasmid isolation was performed and clone was confirmed
by restriction enzyme digestion. The fragment of 2197 bp was obtained by XbaI and XhoI
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digestion and cloned at the same sites in pDrive vector. pDrive was chosen as delivery vector.
It has AmpR, KanR, and oriE for propagation in E. coli. As vector does not have oriM, it works
as a suicide vector in mycobacteria. The generated pDrive:hyg:gadA plasmid was
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electroporated in MS. Transformants were selected on LBGT agar plates with amp and hyg.
Single colonies obtained were replica plated on hyg and kan plates to segregate colonies with
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single crossing over from the cells which had undergone double crossing over event i.e. ΔgadA
mutant cells. Putative mutants screening for double recombinants generating MSΔgadA were
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performed using colony PCR using specific confirmatory primers. One of the mutants was
selected and designated as MSΔgadA and used in the present study. The MSΔgadA generated
was further confirmed by Southern Blotting and Sanger sequencing. A complement strain of
gadA deletion mutant was generated by electroporating pMV361:gadA into MSΔgadA and
selected on kan hyg plates. Integration of gene was confirmed by PCR using vector and gadA
specific primers. One of colony was picked and designated as MSΔgadA::gadA. A complement
of gadA mutant was also generated by electroporating pMV361:gadB into MSΔgadA and
selected on kan hyg plates. Integration of the gene was confirmed by colony PCR using vector
and gadB specific primers. One of colony was picked and designated as MSΔgadA::gadB.

5.5 Southern blotting

The MSΔgadA strain was confirmed by Southern blotting. Equal quantities of Genomic DNA
of MS wild type (MS wt) and putative gadA deletion mutant were digested with using EcoRI
and XhoI. The digested DNAs were and resolved on 0.8% agarose gel and Southern blotting
was performed as described previously for MS with few modifications (39) The probe was
labelled using Dig DNA labelling mix (Sigma-Roche) as per manufacturer’s instructions. Blots
were developed using Dig detection kit according to the manufacturer’s instructions.
5.6 Acid survival assay
Methods as described previously for MS were adopted with few modifications (30, 31).
Mycobacterial cultures were grown until the OD600 reached 0.8-1.0. Equal no. of cells from
each culture were pelleted by centrifugation at 6000 rpm for 10 min. Pellet was then washed
with PBS and then resuspended in LBGT media of either 3.0 ±0.2 pH or 5.0 ±0.2 pH and further
incubated for 4 h and 6 h respectively at 37°C with shaking at 200 rpm. Mycobacterial culture
pellet resuspended in pH 7.0 ±0.2 LBGT media was taken as control under similar conditions.
The viability of cultures was determined using dilution plating which was done before and after
acid challenge on LBGT agar plates. Plates were incubated at 37°C for 3 days. CFUs were
counted and the CFU survival percentage was calculated of each recombinant strain by
comparing CFU of mycobacterial culture plated before and after acid challenge. Survival at
each time point was expressed as a percentage of the 0-hour CFU. The 4 h or 6 h survival
percentage of the wild-type strains was used as the reference (normalized to 1.0), with the
survival rates of the other strains expressed relative to this standard.

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5.7 Intracellular survival assay
Methods as described previously for MS and BCG were adopted with few modifications for
infection (28, 31).

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Preparation of mycobacterial cells for infection
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Mycobacterial cells were grown until the OD600 reached 0.8-1.0. Equal no. of cells from each
culture was pelleted by centrifugation at 6000rpm for 10 min. Pellet was then washed with
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complete DMEM/RPMI medium and resuspended in fresh complete DMEM/RPMI medium.
A single cell bacterial cell suspension was prepared by vortexing the culture with 3 mm sterile
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glass beads five times (2 min each) then suspension was passed through 26-gauge needle
several times to disaggregate any remaining clumps. After homogenization with a needle, the
bacterial suspension was centrifuged for 3 min at 100g to pellet remaining clumps. The total
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no. of bacteria was ascertained by measuring the OD600 of the bacterial suspension and further
by counting the CFUs on LBGT agar/MB7H10-agar plates.
Preparation of mammalian cells for infection
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A total of 1 × 105 THP-1 cells were seeded in 12 well plates and incubated with 50 nM PMA
at 37°C in 5% CO2 for 48 h. A total of 1 × 105 J774.A.1 cells were seeded in 12 well plates and
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incubated for 24 h at 37°C in 5% CO2.

Method for infection
Differentiated THP-1 cells or J774.A.1 macrophage cells were infected with the single-cell
bacterial suspension at a multiplicity of infection (MOI) of 1:10 and incubated for 2 h. To
eliminate extracellular bacteria, amikacin treatment was applied for 2 h, followed by multiple
PBS washes. The cells were lysed with 0.05% SDS, and CFU counts were taken from lysates
to determine intracellular bacterial counts, marking this as the 0-hour time point. For cells
incubated beyond 0 hours, PBS was used to remove residual amikacin, and fresh medium
(without amikacin) was added. The intracellular survival of mycobacterial strains was assessed
using CFU assays at 0 h, 24 h, and 48 h post-infection. Infections with each mycobacterial
strain were set at MOI 1:10, and CFU counts at 0 h served as the baseline (100%) to account
for minor variations in bacterial counts at infection onset. After a 24 h incubation (for MS) or
48 h incubation (for BCG), the macrophages were lysed for CFU determination. Survival at
each time point was expressed as a percentage of the 0-hour CFU. The 24 h survival percentage
of the wild-type strains was used as the reference (normalized to 1.0), with the survival rates
of the other strains expressed relative to this standard.
5.8 Isolation of RNA, cDNA synthesis and quantitative PCR
Methods as described previously for mycobacteria were adopted with few modifications (31,
40). For the gene expression analysis after exposure to acidic medium, RNA from
mycobacterial cultures exposed to acidic medium of pH 5.0 ±0.2 for 4 h was isolated. Cultures
incubated at pH 7.0 ±0.2 were taken as control. For gene expression analysis of mycobacterial
cultures after infection in macrophages, RNA was isolated from mycobacterial cultures
infected within macrophages for 4 h. To isolate RNA from intracellular mycobacteria,
macrophages were subjected to osmotic lysis and released bacteria were pelleted and total RNA
was isolated using TRiZol reagent (Thermo-fisher scientific). Mycobacterial cell lysis was
done using bead beating 5 times each for 45 sec pulse and 60 sec rest. The isolated RNA was
then further purified using RNAeasy purification kit (Qiagen). Purified RNA was quantified
using Nanodrop and to confirm the integrity of RNA, the samples were subjected to agarose
gel electrophoresis. 2 µg of total RNA was digested with RNase free DNase (Ambion) and
used for cDNA synthesis with random hexamer primers using HI capacity reverse cDNA
synthesis kit (Thermo-fisher scientific). Equal amount of cDNA was used in each reaction. To

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confirm absence of genomic DNA in the RNA samples, cDNA reactions without Reverse

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Transcriptase were performed. Reverse Transcriptase Quantitative PCR (qRT-PCR) was
performed in 96 well plates on qTOWER3 G (Analytik jena) using SYBR Green 1 master mix

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(Thermo-fisher scientific) and results were analyzed. Specificity of amplicons was confirmed
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by Melt curve analysis. Further, after the completion, the reactions were subjected to agarose
gel electrophoresis for confirmation of integrity and specificity of reactions. Change in gene
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expression was analyzed by 2-∆∆Ct method. Primer pairs used in the study are listed in
supplementary table 1.
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Acknowledgement
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This study was supported by grants received from SERB, India (Grant ID: CRG/2020/000520)
and MANIT, Bhopal (Grant ID: Dean R&C 19/1021 dated 14-08-2019) both sanctioned to Dr.
Shivendra K. Chaurasiya. Rupal Rai is recipient of Teaching Assistantship from MANIT,
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Bhopal. Lab of Dr. Anirudh K. Singh has been funded by DBT-Ramalingaswami Fellowship
(BT/RLF/Re-Entry/17/2016) and ANRF, Startup research grant, India (Grant ID:
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SRG/2023/001498). We thank Dr. William Jacobs at the Albert Einstein College of Medicine,
USA for providing MC2 6206 strain of Mtb. We sincerely thank Dr. Nisheeth Agarwal at the
Translational Health Science and Technology Institute, Faridabad for providing access to his
laboratory and logistical support to carry out the acid treatment of Mtb.

Competing Interests
The authors have no relevant financial or non-financial interests to disclose.

Data Availability
The datasets generated during and/or analyzed during the current study are available in this
manuscript.

Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
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7. Legends
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Fig. 1 Expression of gad and glutamate metabolism related genes in MS and Mtb in acidic
conditions and during infection in macrophages.
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The relative expression of genes was measured using reverse transcriptase qRT-PCR using
SYBR Green I chemistry. Relative gene expression was determined using the comparative 2-
ΔΔCt
method. A. Effect of acid stress, on gene expression in MS cells grown for 4 h in LBGT
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medium, pH 5.0±0.2. The relative expression of the target genes in acidic medium was
compared with MS cells grown in LBGT medium, pH 7.0±0.2. 16SrRNA was used as the
internal reference gene for normalization of the expression data. B. Expression of the target
genes in MS cells during infection in J774.A.1 cells for 4 h at MOI 10:1. A portion of MS cells
used for the infection were taken as control, sigA was used as the internal reference gene to
normalize the expression data. C. The relative expression of target genes in Mtb cells grown in
MB7H9 medium, pH 5.0±0.2. Cells grown in MB7H9, pH 7.0±0.2 were used as control and
16S rRNA was used as the internal reference for normalizing the expression data. D. The
relative expression of target genes in Mtb cells during infection in THP1 cells for 4 h at MOI
10:1. A portion of Mtb cells used for the infection served as control and 16S rRNA was used as
the internal reference to normalize the expression data. Each experiment was done at three
independent occasions in triplicates and the data represent the mean ± SD. (*p < 0.05, **p <
0.005, ns p > 0.0.5; one-tailed Student’s t-test using GraphPad Prism software)

Fig. 2. Ectopic overexpression of gad supports acid tolerance in mycobacteria.

A. Confirmation of the integration of gadA and gadB in the genome of MS by colony PCR. M-
DNA ladder 1. MS::gadA (1566 bp); 2. MS::gadB (1569 bp); 3. MS:EV. B. Detection of Gad
activity in MS recombinants overexpressing Gad. Mycobacterial cultures at OD600-0.6-0.8
were harvested by centrifugation, washed with PBS and resuspended in Gad reagent. The Gad
overexpressing MS strain produced blue colour within 30 min of incubation. 1. MS::gadA; 2.
MS::EV; 3. MS::gadB. C. Ectopic overexpression of gad in MS confers higher acid resistance
at a lethal pH 3.0. MS recombinants were cultured at OD600-0.6-0.8 and were harvested by
centrifugation, washed with PBS and resuspended in pH 3.0 ±0.2 and further incubated for 4 h
at 37°C, 200 rpm. The values represent relative survival of MS::gadA and MS::gadB compared
with MS::EV separately (*p -0.0229, ** p -0.0470; two-tailed Student’s t-test using GraphPad
Prism software). The data represent the mean ± SD from three independent experiments, each
performed in triplicates.

Fig. 3 Generation of MS gadA deletion mutant

A. A Schematic of allelic exchange cassette for the gadA deletion in MS. B. Southern
Hybridization. DNA of MS wt and MSΔgadA were digested with EcoRI and XhoI and
transferred to a Nylon membrane and probed with a digoxygenin labelled probe
complementary to gadA and its upstream sequence 1. MS wt DNA, fragment of 5664 bp
confirms the wt; 2. MSΔgadA DNA, fragment of 2569 bp confirms the deletion mutant. C. Gad

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assay to confirm the loss of Gad activity in MSΔgadA strain and restoration by

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complementation. Mycobacterial cultures at OD600-0.6-0.8 were harvested by centrifugation,
washed with PBS and resuspended in Gad reagent. Cultures producing blue colour within 4 h

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were considered positive.
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Fig. 4 Deletion of gadA reduces survival of MS in acidic medium
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A. gadA in MS confers higher acid resistance at a lethal pH 3.0. MS recombinants were cultured
at OD600-0.6-0.8 and were harvested by centrifugation, washed with PBS and resuspended in
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pH 3.0 ±0.2 and further incubated for 4 h at 37°C, 200rpm. The values represent relative
survival of each strain compared with MS wt (*p -0.0226, ** p -0.0057, ***p -0.028; two-tailed
Student’s t-test using GraphPad Prism software). Error bars represent standard deviations from
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three independent experiments performed in triplicates. B. gadA in MS supports survival at a

sub lethal pH 5.0. MS recombinants were cultured at OD600-0.6-0.8 and were harvested by
centrifugation, washed with PBS and resuspended in pH 5.0 ±0.2 and further incubated for 6 h
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at 37°C, 200 rpm. The values represent the relative survival of each strain compared with MS
wt (*p -0.02, ns p -0.3750; two-tailed Student’s t-test using GraphPad Prism software). The
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data represent the mean ± SD from three independent experiments performed in triplicates.

Fig. 5 Gad promotes intracellular survival of mycobacteria within macrophages

A. Intracellular survival of recombinant MS in THP-1 cells. Mycobacterial cultures were
grown in normal LBGT medium, pelleted and resuspended complete RPMI, single cell
suspension was prepared and infected in THP-1 cells with MOI 10:1. The values represent
relative survival of MSΔgadA when compared to MS wt after 24 h of infection (*p -0.0045,
two-tailed Student’s t-test using GraphPad Prism software). The data represent the mean ± SD
from three independent experiments performed in triplicates. B. Intracellular survival of
recombinant MS in J774.A.1 cells. Mycobacterial cultures were grown in normal LBGT
medium, pelleted and resuspended in complete DMEM, single cell suspension was prepared
and infected in differentiated J774.A.1 cells with MOI 10:1. The values represent relative
survival of MSΔgadA when compared to MS wt after 24 h of infection (*p -0.0417, two-tailed
Student’s t-test using GraphPad Prism software). Data represent the mean ± SD from three
independent experiments performed in triplicates. C. Cloning of gadB in BCG. M – DNA
ladder; 1. BCG::EV; 2. BCG::gadB, DNA fragment of size 1569 bp confirms the integration
of gene. D. Detection of Gad activity in Gad overexpressing BCG strain. Mycobacterial
cultures at OD600-0.6-0.8 were harvested by centrifugation, washed with PBS and resuspended
in Gad reagent. Cultures producing blue colour within 4 h were considered positive. 1.
BCG::EV; 2. BCG::gadB. E. Intracellular survival of BCG overexpressing gad in J774.A.1
cells. Mycobacterial cultures were grown in 7H9 medium, pelleted and resuspended complete
DMEM, single cell suspension was prepared and infected in J774.A.1 cells. The values
represent relative survival of BCG::gadB when compared to BCG::EV after 48 h of infection
(*p -0.0331, two-tailed Student’s t-test using GraphPad Prism software). The data represent the
mean ± SD from three independent experiments performed in triplicates.

Fig. 6 Proposed mechanism of glutamate decarboxylase (Gad)-mediated acid resistance

in mycobacteria
The decarboxylation reaction catalyzed by Gad consumes a proton, helping to neutralize the
intracellular acidity during acidic stress. Since the mycobacterial genome does not encode a
glutamate-GABA antiporter, sustained glutamate decarboxylation relies on the availability of
glutamate and the removal of GABA. Mtb lacks Kdh activity, preventing the conversion of α-
ketoglutarate to succinyl-CoA (46). Instead, kdh encodes α-ketoglutarate decarboxylase, which
converts α-ketoglutarate into succinic semialdehyde (SSA). Reduced expression of α-

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ketoglutarate decarboxylase may inhibit this conversion, potentially stalling the TCA cycle.
Under these conditions, GABA produced by Gad can react with α-ketoglutarate via the GABA

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shunt, generating SSA and glutamate. This process restores TCA cycle activity and regenerates
-p
glutamate, enabling continuous decarboxylation and intracellular pH regulation.
Genes involved in glutamate metabolism (gdh, glnA1, gltD/gltB) likely contribute to
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maintaining intracellular glutamate levels during acidic stress. The upregulation of gltD/B and
downregulation of gdh and glnA1 suggest a metabolic shift toward glutamate synthesis to
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sustain the glutamate pool.

The upregulation of fadD9, encoding fatty acyl-CoA synthetase, may help maintain the carbon
pool by channelling acetyl-CoA into the TCA cycle. When carbon is derived from acetyl-CoA
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through fatty acid oxidation, coupling TCA activity with glutamate decarboxylation may
support acid tolerance by facilitating fatty acid oxidation.
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Under these conditions, acetyl-CoA provides the carbon input, while CO₂ released from
glutamate decarboxylation serves as the output, preserving glutamate for other critical
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functions. The upregulation of icl and gab-T further highlights the importance of alternative
TCA pathways in sustaining metabolism under acidic stress.
Color Codes:
Green: Genes encoding enzymes upregulated at acidic pH in Mtb.
Red: Genes encoding enzymes downregulated at acidic pH in Mtb.

Abbreviations
TB - Tuberculosis
Mtb - Mycobacterium tuberculosis
MDR - multidrug-resistant
XDR - extensively drug-resistant
TDR - totally drug-resistant
V-ATPase - vacuolar proton-ATPase
PknG - Protein kinase G
Gad - glutamate decarboxylase
MS - Mycobacterium smegmatis
LB - Luria-Bertani
LBGT - LB with 0.5% glycerol and 0.05% tween 80
ADC - albumin-dextrose-catalase
OADS - 1× oleic acid-albumin-dextrose-saline
E.coli - Escherichia coli
kan - kanamycin
amp - ampicillin
hyg - hygromycin
NCCS - National Centre of Cell Science
PMA - Phorbol 12- Myristate, 13-acetate
EV - empty vector
PBS - phosphate buffer saline
wt - wild type
qRT-PCR - Quantitative real time PCR
gaba-at - GABA-aminotransferase
gdh - Glutamate dehydrogenase

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fadD9 - Fatty acyl CoA synthetase

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gltD - Glutamate synthase
sucA - 2-oxoglutarate dehydrogenase

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glnA1 - glutamine synthetase
gltD6468 - Glutamate synthase -p
gabD2 - Succinic semialdehyde dehydrogenase
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icl1 - Isocitratelyase
kdh - α-Keto-glutarate Dehydrogenase
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TCA - tricarboxylic acid

SSA - succinic semialdehyde
TraDIS - transposon-directed insertion site sequencing
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CDRI - Central Drug Research Institute

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BJM - Formal analysis, Methodology
RC - Conceptualization, Methodology, Writing – review & editing.
AKS - Resources, Methodology, Writing – review & editing.
SKC - Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology,
Project administration, Resources, Supervision, Validation, Visualization, Writing – review &
editing.

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