Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosis. D. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis. Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

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Nickel Promotes Biofilm Formation by Escherichia coli K-12 Strains That Produce Curli

Applied and Environmental Microbiology ◽

10.1128/aem.02171-08 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1723-1733 ◽

Cited By ~ 40

Author(s):

Claire Perrin ◽

Romain Briandet ◽

Gregory Jubelin ◽

Philippe Lejeune ◽

Marie-Andrée Mandrand-Berthelot ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Selective Advantage ◽

Bacterial Survival ◽

Toxic Compounds ◽

E Coli ◽

Transcriptional Effect ◽

K 12 ◽

Encoding Genes

ABSTRACT The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.

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Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance

Journal of Bacteriology ◽

10.1128/jb.181.4.1343-1347.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1343-1347 ◽

Cited By ~ 89

Author(s):

Mark Payton ◽

Roy Auty ◽

Rupika Delgoda ◽

Martin Everett ◽

Edith Sim

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Antitubercular Drug ◽

Isoniazid Resistance ◽

E Coli ◽

Monospecific Antiserum ◽

Many Sources ◽

Eukaryotic Organisms

ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

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The Three RelE Homologs of Mycobacterium tuberculosis Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance

Journal of Bacteriology ◽

10.1128/jb.01285-09 ◽

2010 ◽

Vol 192 (5) ◽

pp. 1279-1291 ◽

Cited By ~ 81

Author(s):

Ramandeep Singh ◽

Clifton E. Barry ◽

Helena I. M. Boshoff

Keyword(s):

Mycobacterium Tuberculosis ◽

Survival Rates ◽

Specific Effect ◽

Multidrug Resistant ◽

Cross Resistance ◽

Bacterial Survival ◽

E Coli ◽

The Face

ABSTRACT In Escherichia coli, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of E. coli, persisters of Mycobacterium tuberculosis selected with one drug do not acquire cross-resistance to other classes of drugs. M. tuberculosis has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both M. tuberculosis and E. coli, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated in vivo, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin in vitro, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that, in vivo, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant M. tuberculosis.

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MsDpo4—a DinB Homolog fromMycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

Journal of Nucleic Acids ◽

10.1155/2012/285481 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Amit Sharma ◽

Deepak T. Nair

Keyword(s):

Mycobacterium Tuberculosis ◽

Dna Polymerase ◽

Environmental Conditions ◽

Mycobacterium Smegmatis ◽

Molecular Level ◽

Dna Polymerases ◽

Adaptive Mutagenesis ◽

E Coli ◽

Nucleotide Incorporation ◽

Y Family

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of thedinBgene in case ofE. coli. However, unlikeE. coli, it has been seen that expression of the homologs ofdinBinMycobacterium tuberculosisare not upregulated under conditions of stress. These studies suggest that DinB homologs inMycobacteriamight not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog fromMycobacterium smegmatis(MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.

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Expression and purification of immunologically reactive DPPD, a recombinantMycobacterium tuberculosisskin test antigen, usingMycobacterium smegmatisandEscherichia colihost cells

Canadian Journal of Microbiology ◽

10.1139/w03-109 ◽

2004 ◽

Vol 50 (2) ◽

pp. 97-105 ◽

Cited By ~ 5

Author(s):

Chao Liu ◽

Eric Flamoe ◽

Hong-Jing Chen ◽

Darrick Carter ◽

Steven G Reed ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Guinea Pigs ◽

Plasmid Vector ◽

Host Cells ◽

Delayed Type Hypersensitivity ◽

Hypersensitivity Reactions ◽

Expression And Purification ◽

Purified Protein Derivative ◽

E Coli

DPPD is a Mycobacterium tuberculosis recombinant antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both guinea pigs and humans infected with M. tuberculosis. In addition, earlier clinical studies with DPPD suggested that this molecule could improve the specificity of the tuberculin skin test, which is used as an important aid for the diagnosis of tuberculosis. However, these studies could only be performed with DPPD engineered as a fusion molecule with another Mycobacterium spp. protein because no expression of DPPD could be achieved as a single molecule or as a conventional fusion protein in any commercial system. Although recombinant fusion proteins are in general suitable for several biological studies, they are by definition not ideal for studies involving highly purified and defined polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive DPPD in M. smegmatis could be achieved. More abundant expression and purification of DPPD as a homo-trimer molecule was achieved in E. coli ([Formula: see text]2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the DPPD gene containing its leader peptide. However, the expressed proteins lacked the leader sequence, which indicates that processing of the M. tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M. tuberculosis were indistinguishable from that elicited by purified protein derivative. Because the DPPD gene is present only in the tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.Key words: Mycobacterium tuberculosis, Mycobacterium smegmatis skin test, DTH, DPPD.

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Mycobacterium tuberculosis LprE enhances bacterial persistence by inhibiting cathelicidin and autophagy in macrophages

10.1101/230698 ◽

2017 ◽

Author(s):

Avinash Padhi ◽

Ella Bhagyaraj ◽

Mehak Zahoor Khan ◽

Mainak Biswas ◽

Srabasti Sengupta ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Mycobacterium Tuberculosis ◽

Immune Responses ◽

Signaling Pathway ◽

Mycobacterium Smegmatis ◽

Bacterial Survival ◽

Bacterial Persistence ◽

Host Immune Responses ◽

Mycobacterial Pathogenesis

ABSTRACTMycobacterium tuberculosis(Mtb)lipoproteins are known to facilitate bacterial survival by manipulating the host immune responses. Here, we have characterized a novelMtblipoprotein LprE(LprEMtb), and demonstrated its role in mycobacterial survival. LprEMtbacts by down-regulating the expression of cathelicidin, Cyp27B1, VDR and p38-MAPK via TLR-2 signaling pathway. Deletion oflprEMtbresulted in induction of cathelicidin and decreased survival in the host. Interestingly, LprEMtbwas also found to inhibit autophagy mechanism to dampen host immune response. Episomal expression of LprEMtbin non-pathogenicMycobacterium smegmatis(Msm) increased bacillary persistence by down-regulating the expression of cathelicidin and autophagy, while deletion of LprEMtborthologue inMsm, had no effect on cathelicidin and autophagy expression. Moreover, LprEMtbblocked phago-lysosome fusion by suppressing the expression of EEA1, Rab7 and LAMP-1 endosomal markers by down-regulating IL-12 and IL-22 cytokines. Our results indicate that LprEMtbplays an important role in mycobacterial pathogenesis in the context of innate immunity.

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Identification and Analysis of “Extended −10” Promoters from Mycobacteria

Journal of Bacteriology ◽

10.1128/jb.180.9.2568-2573.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2568-2573 ◽

Cited By ~ 42

Author(s):

Murali D. Bashyam ◽

Anil K. Tyagi

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Sigma Factor ◽

Mycobacterium Smegmatis ◽

Important Determinant ◽

Comparative Assessment ◽

Binding Domain ◽

Site Specific ◽

E Coli

ABSTRACT Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. colicounterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosispromoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.

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Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

10.1101/825588 ◽

2019 ◽

Author(s):

Farah Shamma ◽

Kadamba Papavinasasundaram ◽

Samantha Y. Quintanilla ◽

Aditya Bandekar ◽

Christopher Sassetti ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Control Cell ◽

Model Organism ◽

Mycolic Acid ◽

Regulatory Function ◽

Antibiotic Tolerance ◽

Bacterial Survival ◽

Cell Wall Metabolism

AbstractMycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

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Regulation of Expression of mas and fadD28, Two Genes Involved in Production of Dimycocerosyl Phthiocerol, a Virulence Factor of Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.184.24.6796-6802.2002 ◽

2002 ◽

Vol 184 (24) ◽

pp. 6796-6802 ◽

Cited By ~ 17

Author(s):

Tatiana D. Sirakova ◽

Ann Marie Fitzmaurice ◽

Pappachan Kolattukudy

Keyword(s):

Mycobacterium Tuberculosis ◽

Virulence Factor ◽

Mycobacterium Smegmatis ◽

Transcriptional Enhancer ◽

Regulation Of Expression ◽

Gene Encoding ◽

E Coli ◽

Translational Start ◽

High Level ◽

Acyl Coenzyme A

ABSTRACT Transcriptional regulation of genes involved in the biosynthesis of cell wall lipids of Mycobacterium tuberculosis is poorly understood. The gene encoding mycocerosic acid synthase (mas) and fadD28, an adjoining acyl coenzyme A synthase gene, involved in the production of a virulence factor, dimycocerosyl phthiocerol, were cloned from Mycobacterium bovis BCG, and their promoters were analyzed. The putative promoters were fused to the xylE reporter gene, and its expression was measured in Escherichia coli, Mycobacterium smegmatis, and M. bovis BCG. In E. coli, the fadD28 promoter was not functional but the mas promoter was functional. Both fadD28 and mas promoters were functional in M. smegmatis, at approximately two- and sixfold-higher levels, respectively, than the BCG hsp60 promoter. In M. bovis BCG, the fadD28 and mas promoters were functional at three- and fivefold-higher levels, respectively, than the hsp60 promoter. Primer extension analyses identified transcriptional start points 60 and 182 bp upstream of the translational start codons of fadD28 and mas, respectively. Both promoters contain sequences similar to the canonical −10 and −35 hexamers recognized by the σ70 subunit of RNA polymerase. Deletions of the upstream regions of both genes indicated that 324 bp of the fadD28 and 228 bp of the mas were essential for promoter activity. Further analysis of the mas promoter showed that a 213-bp region 581 bp upstream of the mas promoter acted as a putative transcriptional enhancer, promoting high-level expression of the mas gene when present in either direction. This represents the identification of a rare example of an enhancer-like element in mycobacteria.

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Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00563-20 ◽

2020 ◽

Author(s):

Farah Shamma ◽

Kadamba Papavinasasundaram ◽

Samantha Y. Quintanilla ◽

Aditya Bandekar ◽

Christopher Sassetti ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Control Cell ◽

Model Organism ◽

Mycolic Acid ◽

Regulatory Function ◽

Antibiotic Tolerance ◽

Bacterial Survival ◽

Cell Wall Metabolism

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. Importance Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

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