The Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor SigL Regulates Polyketide Synthases and Secreted or Membrane Proteins and Is Required for Virulence

ABSTRACT Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the −35 region and CGTgtc in the −10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.

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Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00687-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5472-5479 ◽

Cited By ~ 8

Author(s):

Ruben C. Hartkoorn ◽

Claudia Sala ◽

Sophie J. Magnet ◽

Jeffrey M. Chen ◽

Florence Pojer ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Stationary Phase ◽

Sigma Factor ◽

Potent Inhibitor ◽

Sigma Factors ◽

Antituberculosis Drugs ◽

Axenic Cultures

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

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Examination of Mycobacterium tuberculosis sigma factor mutants using low-dose aerosol infection of guinea pigs suggests a role for SigC in pathogenesis

Microbiology ◽

10.1099/mic.0.28591-0 ◽

2006 ◽

Vol 152 (6) ◽

pp. 1591-1600 ◽

Cited By ~ 39

Author(s):

Russell K. Karls ◽

Jeannette Guarner ◽

David N. McMurray ◽

Kristin A. Birkness ◽

Frederick D. Quinn

Keyword(s):

Mycobacterium Tuberculosis ◽

Sigma Factor ◽

Guinea Pigs ◽

Low Dose ◽

Sigma Factors ◽

Respiratory Pathogen ◽

Growth Deficiency ◽

Aerosol Infection

Secondary sigma factors in bacteria direct transcription of defence regulons in response to specific stresses. To identify which sigma factors in the human respiratory pathogen Mycobacterium tuberculosis are important for adaptive survival in vivo, defined null mutations were created in individual sigma factor genes. In this study, in vitro growth virulence and guinea pig pathology of M. tuberculosis mutants lacking functional sigma factors (SigC, SigF, or SigM) were compared to the parent strain, H37Rv. None of the mutant strains exhibited a growth deficiency in Middlebrook 7H9 broth, nor were any impaired for intracellular replication in the human monocytic macrophage cell-line THP-1. Following low-dose aerosol infection of guinea pigs, however, differences could be detected. While a SigM mutant resulted in lung and spleen granulomas of comparable composition to those found in H37Rv-infected animals, a SigF mutant was partially attenuated, exhibiting necrotic spleen granulomas and ill-defined lung granulomas. SigC mutants exhibited attenuation in the lung and spleen; notably, necrotic granulomas were absent. These data suggest that while SigF may be important for survival in the lung, SigC is likely a key regulator of pathogenesis and adaptive survival in the lung and spleen. Understanding how SigC mediates survival in the host should prove useful in the development of anti-tuberculosis therapies.

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Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro

Infection and Immunity ◽

10.1128/iai.73.5.3038-3043.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 3038-3043 ◽

Cited By ~ 117

Author(s):

Katrina J. Downing ◽

Vladimir V. Mischenko ◽

Margarita O. Shleeva ◽

Danielle I. Young ◽

Michael Young ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Micrococcus Luteus ◽

Most Probable Number ◽

Infection Model ◽

Term Survival ◽

Probable Number ◽

Mouse Infection Model ◽

Nonculturable State

ABSTRACT Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the ΔrpfB, ΔrpfD, and ΔrpfE strains). In contrast, two multiple strains, KDT8 (ΔrpfA-mutation ΔrpfC ΔrpfB) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

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ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

Journal of Bacteriology ◽

10.1128/jb.182.16.4606-4616.2000 ◽

2000 ◽

Vol 182 (16) ◽

pp. 4606-4616 ◽

Cited By ~ 96

Author(s):

Maureen J. Bibb ◽

Virginie Molle ◽

Mark J. Buttner

Keyword(s):

Rna Polymerase ◽

Sigma Factor ◽

Streptomyces Coelicolor ◽

Aerial Mycelium ◽

Sigma Factors ◽

Colony Development ◽

Null Mutant ◽

Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

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An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906606116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 15907-15913 ◽

Cited By ~ 11

Author(s):

Hua Wang ◽

Alexander A. Fedorov ◽

Elena V. Fedorov ◽

Debbie M. Hunt ◽

Angela Rodgers ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Resistance Mechanism ◽

Infection Model ◽

Enzymatic Assays ◽

X Ray Crystallography ◽

Mouse Infection Model ◽

Leucine Catabolism ◽

Global Population

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

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The Heat-Induced Molecular Disaggregase Hsp104 of Candida albicans Plays a Role in Biofilm Formation and Pathogenicity in a Worm Infection Model

Eukaryotic Cell ◽

10.1128/ec.00147-12 ◽

2012 ◽

Vol 11 (8) ◽

pp. 1012-1020 ◽

Cited By ~ 17

Author(s):

Alessandro Fiori ◽

Soňa Kucharíková ◽

Gilmer Govaert ◽

Bruno P. A. Cammue ◽

Karin Thevissen ◽

...

Keyword(s):

Candida Albicans ◽

Stress Responses ◽

Biofilm Formation ◽

Fungal Pathogens ◽

Elevated Temperatures ◽

Structural Defects ◽

Infection Model ◽

Content Type

ABSTRACT The consequences of deprivation of the molecular chaperone Hsp104 in the fungal pathogen Candida albicans were investigated. Mutants lacking HSP104 became hypersusceptible to lethally high temperatures, similarly to the corresponding mutants of Saccharomyces cerevisiae , whereas normal susceptibility was restored upon reintroduction of the gene. By use of a strain whose only copy of HSP104 is an ectopic gene under the control of a tetracycline-regulated promoter, expression of Hsp104 prior to the administration of heat shock could be demonstrated to be sufficient to confer protection from the subsequent temperature increase. This result points to a key role for Hsp104 in orchestrating the cell response to elevated temperatures. Despite their not showing evident growth or morphological defects, biofilm formation by cells lacking HSP104 proved to be defective in two established in vitro models that use polystyrene and polyurethane as the substrates. Biofilms formed by the wild-type and HSP104 -reconstituted strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the hsp104 Δ/ hsp104 Δ mutant showed structural defects and appeared patchy and loose. Decreased virulence of the hsp104 Δ/ hsp104 Δ mutant was observed in the Caenorhabditis elegans infection model, in which high in vivo temperature does not play a role. In agreement with the view that stress responses in fungal pathogens may have evolved to provide niche-specific adaptation to environmental conditions, these results provide an indication of a temperature-independent role for Hsp104 in support of Candida albicans virulence, in addition to its key role in governing thermotolerance.

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Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2294-2301.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2294-2301 ◽

Cited By ~ 207

Author(s):

Anne J. Lenaerts ◽

Veronica Gruppo ◽

Karen S. Marietta ◽

Christine M. Johnson ◽

Diane K. Driscoll ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Load ◽

Multidrug Resistant ◽

Infection Model ◽

Significant Activity ◽

Dependent Manner ◽

In Vivo Models ◽

Long Term Treatment

ABSTRACT This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

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Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia

Microbiology ◽

10.1099/mic.0.047381-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1816-1822 ◽

Cited By ~ 9

Author(s):

Samuele Peppoloni ◽

Brunella Posteraro ◽

Bruna Colombari ◽

Lidia Manca ◽

Axel Hartke ◽

...

Keyword(s):

Superoxide Dismutase ◽

Enterococcus Faecalis ◽

Stress Responses ◽

Peritoneal Macrophages ◽

Infection Process ◽

Microglial Cells ◽

Infection Model

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo–in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

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Blocking the alternative sigma factor RpoN reduces virulence ofPseudomonas aeruginosaisolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

10.1101/340307 ◽

2018 ◽

Author(s):

MG Lloyd ◽

JL Vossler ◽

CT Nomura ◽

JF Moffat

Keyword(s):

Cystic Fibrosis ◽

Sigma Factor ◽

Antimicrobial Agents ◽

Laboratory Strain ◽

Infection Model ◽

Alternative Sigma Factor ◽

Beta Lactam ◽

Cis Acting

AbstractMultidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria.Pseudomonas aeruginosais an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections.P. aeruginosais inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment.P. aeruginosahas virulence factors that are regulated by sigma factors in response to the tissue microenvironment. The alternative sigma factor, RpoN (σ54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a “molecular roadblock”, binding RpoN consensus promoters at the −24 site and blocking transcription. RpoN* reduces virulence ofP. aeruginosalaboratory strains bothin vitroandin vivo,but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically variedP. aeruginosastrains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in aP. aeruginosa – C. elegansinfection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strainP. aeruginosaPA19660Xen5. Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

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A Complex Network of Sigma Factors and sRNA StsR Regulates Stress Responses in R. sphaeroides

International Journal of Molecular Sciences ◽

10.3390/ijms22147557 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7557

Author(s):

Katrin M. H. Eisenhardt ◽

Bernhardt Remes ◽

Julian Grützner ◽

Daniel-Timon Spanka ◽

Andreas Jäger ◽

...

Keyword(s):

Stress Responses ◽

Sigma Factors ◽

Photooxidative Stress ◽

Alternative Sigma Factors ◽

Special Challenge ◽

Photosynthesis Genes ◽

In Vitro Interaction ◽

Complex Regulatory Network

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.

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