Malate:quinone oxidoreductase knockout makes Mycobacterium tuberculosis susceptible to stress and affects its in vivo survival.

Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_2875, annotated as malate:quinone oxidoreductase (mqo), is thought to have a role in TCA cycle in converting malate to oxaloacetate. To study its physiological relevance, we developed mqo knockout (KO) in Mtb-Ra. A KO complemented (KOC) strain was also developed by complementing the KO with mqo over-expressing construct. Under normal in vitro conditions, KO does not show any growth defect but showed reduced CFU burden in macrophages and in mice lungs. In vitro studies with KO showed reduced fitness under oxidative and low pH stress, and also increased susceptibility to levofloxacin and D-cycloserine. Transcript analysis of mqo showed increased expression levels under oxidative and low pH stress. This is the first study to show physiological relevance of mqo encoded by MRA_2875 in Mtb-Ra under oxidative and low pH stress. In summary, the present study shows that MRA_2875 encoded malate:quinone oxidoreductase is a functional enzyme which contributes to oxidative stress and low pH tolerance, and survival in macrophages and in mice.

GlfT1 down-regulation affects Mycobacterium tuberculosis biofilm formation and its in-vitro and in-vivo survival.

Mycobacterial galactan biosynthesis is critical for cell viability and growth, therefore an effort was made to study galactofuranosyl transferase 1, encoded by MRA_3822 in Mycobacterium tuberculosis H37Ra (Mtb-Ra). Galactofuranosyl transferases are involved in the biosynthesis of mycobacterial cell wall galactan chain and have been shown to be essential for in-vitro growth of Mycobacterium tuberculosis. In Mtb-Ra and Mycobacterium tuberculosis H37Rv (Mtb-Rv), two galactofuranosyl transferases are present, GlfT1 acts as initiator of galactan biosynthesis and GlfT2 continues with the subsequent polymerization events. GlfT2 has been well studied however GlfT1 inhibition/down-regulation and its effect on mycobacterial survival fitness has not been evaluated. To study the Mtb-Ra survival after GlfT1 silencing, Mtb-Ra knockdown and complemented strains were developed. In this study we show that GlfT1 down-regulation leads to increased susceptibility to ethambutol. Expression of glfT1 was up-regulated in the presence of ethambutol, and also in the presence of oxidative and nitrosative stress and upon exposure to low pH. Also, reduced biofilm formation, increased accumulation of ethidium bromide, and reduced tolerance to peroxide, nitric oxide and acid stress, were observed. The present study also demonstrates that GlfT1 down-regulation leads to reduced survival of Mtb-Ra in macrophages and in mice.

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice.

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.

Mycobacterium tuberculosis survival and biofilm formation studies: effect of D-amino acids, D-cycloserine and its components.

D-amino acids play an important role in cell wall peptidoglycan biosynthesis. Mycobacterium tuberculosis D-amino acid oxidase deletion led to reduced biofilm-forming ability. Other recent studies also suggest that the accumulation of D-amino acids blocks biofilm formation and could also disperse pre-formed biofilm. Biofilms are communities of bacterial cells protected by extracellular matrix and harbor drug-tolerant as well as persistent bacteria. In Mycobacterium tuberculosis, biofilm formation or its inhibition by D-amino acids is yet to be tested. In the present study, we used selected D-amino acids to study their role in the prevention of biofilm formation and also if D-cycloserine's activity was due to presence of D-Serine as a metabolite. It was observed that D-serine limits biofilm formation in Mycobacterium tuberculosis H37Ra (Mtb-Ra), but it shows no effect on pre-formed biofilm. Also, D-cycloserine and its metabolic product, hydroxylamine, individually and in combination, with D-Serine, limit biofilm formation in Mtb-Ra and also disrupts existing biofilm. In summary, we demonstrated that D-alanine, D-valine, D-phenylalanine, D-serine, and D-threonine had no disruptive effect on pre-formed biofilm of Mtb-Ra, either individually or in combination, and D-cycloserine and its metabolite hydroxylamine have potent anti-biofilm activity.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase.

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli. Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Knockout of MRA_1916 in Mycobacterium tuberculosis H37Ra affects its growth, biofilm formation, survival in macrophages and in mice.

Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_1916 is annotated as a D-amino acid oxidase (DAO). These enzymes perform conversion of d-amino acids to corresponding imino acids followed by conversion into α-keto-acids. In the present study Mtb-Ra recombinants with DAO knockout (KO) and knockout complemented with DAO over-expressing plasmid (KOC) were constructed. The growth studies showed loss of growth of KO in medium containing glycerol as a primary carbon source. Substituting glycerol with acetate or with FBS addition, restored the growth. Growth was also restored in complemented strain (KOC). KO showed increased permeability to hydrophilic dye EtBr and reduced biofilm formation. Also, its survival in macrophages was low. Phagosome maturation studies suggested enhanced colocalization of KO, compared to WT, with lysosomal marker cathepsin D. Also, an increased intensity of Rab5 and iNOS was observed in macrophages infected with KO, compared to WT and KOC. The in vivo survival studies showed no increase in CFU of KO. This is the first study to show functional relevance of DAO encoded by MRA_1916 for Mtb-Ra growth on glycerol, its permeability and biofilm formation. Also, this study clearly demonstrates that DAO deletion leads to Mtb-Ra failing to grow in macrophages and in mice.