scholarly journals Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

2003 ◽  
Vol 71 (7) ◽  
pp. 3927-3936 ◽  
Author(s):  
Michael V. Tullius ◽  
Günter Harth ◽  
Marcus A. Horwitz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Guinea Pigs ◽  
Type Strain ◽  
Wild Type Strain ◽  
Broth Culture ◽  
Wild Type ◽  
Human Macrophages ◽  
Serovar Typhimurium ◽  
Guinea Pig Model

ABSTRACT To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for l-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of l-glutamine, the effective concentration of l-glutamine in the M. tuberculosis phagosome of THP-1 cells was ∼10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruiqi Wang ◽  
Kun Li ◽  
Jifang Yu ◽  
Jiaoyu Deng ◽  
Yaokai Chen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Clinical Isolates ◽  
Aminobenzoic Acid ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Expression Levels ◽  
Encoding Gene ◽  
Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals A Plasmid-Encoded FetMP-Fls Iron Uptake System Confers Selective Advantages to Salmonella enterica Serovar Typhimurium in Growth under Iron-Restricted Conditions and for Infection of Mammalian Host Cells

2020 ◽  
Vol 8 (5) ◽  
pp. 630
Author(s):  
Vanesa García ◽  
Ana Herrero-Fresno ◽  
Rosaura Rodicio ◽  
Alfonso Felipe-López ◽  
Ignacio Montero ◽  
...  
Keyword(s):  
Iron Content ◽  
Salmonella Enterica ◽  
Iron Uptake ◽  
Type Strain ◽  
Wild Type Strain ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Iron Acquisition ◽  
Clinical Strain ◽  
Wild Type ◽  
Serovar Typhimurium

The resistance plasmid pUO-StVR2, derived from virulence plasmid pSLT, is widespread in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain and other European countries. pUO-StVR2 carries several genes encoding a FetMP-Fls system, which could be involved in iron uptake. We therefore analyzed S. Typhimurium LSP 146/02, a clinical strain selected as representative of the isolates carrying the plasmid, and an otherwise isogenic mutant lacking four genes (fetMP-flsDA) of the fetMP-fls region. Growth curves and determination of the intracellular iron content under iron-restricted conditions demonstrated that deletion of these genes impairs iron acquisition. Thus, under these conditions, the mutant grew significantly worse than the wild-type strain, its iron content was significantly lower, and it was outcompeted by the wild-type strain in competition assays. Importantly, the strain lacking the fetMP-flsDA genes was less invasive in cultured epithelial HeLa cells and replicated poorly upon infection of RAW264.7 macrophages. The genes were introduced into S. Typhimurium ATCC 14028, which lacks the FetMP-Fls system, and this resulted in increased growth under iron limitation as well as an increased ability to multiply inside macrophages. These findings indicate that the FetMP-Fls iron acquisition system exceeds the benefits conferred by the other high-affinity iron uptake systems carried by ATCC 14028 and LSP 146/02. We proposed that effective iron acquisition by this system in conjunction with antimicrobial resistance encoded from the same plasmid have greatly contributed to the epidemic success of S. Typhimurium isolates harboring pUO-StVR2.

scholarly journals Sigma B Contributes to Listeria monocytogenes Gastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model

2006 ◽  
Vol 74 (2) ◽  
pp. 876-886 ◽  
Author(s):  
M. R. Garner ◽  
B. L. Njaa ◽  
M. Wiedmann ◽  
K. J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Type Strain ◽  
Wild Type Strain ◽  
Critical Role ◽  
Host Cells ◽  
Gastrointestinal Infection ◽  
Wild Type ◽  
Systemic Spread

ABSTRACT Contributions of the alternative sigma factor σB to Listeria monocytogenes infection were investigated using strains bearing null mutations in sigB, prfA, or inlA or in selected inlA or prfA promoter regions. The ΔP4 inlA strain, which has a deletion in the σB-dependent P4 inlA promoter, and the ΔsigB strain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σB dependent P2 prfA promoter region did not differ from that of the wild type. The virulence of the ΔsigB and ΔP4 inlA strains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigB strain showing greater attenuation, while the virulence capacity of the ΔP2 prfA strain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigB mutation does not result from loss of σB-dependent prfA transcription. Our results show that σB-dependent activation of inlA is important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition to inlA appear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigB strain was not attenuated in intravenously infected guinea pigs. We conclude that (i) L. monocytogenes σB plays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects on inlA transcription but not on prfA transcription, and (iii) σB plays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.

scholarly journals Oral Immunization with an rfaH Mutant Elicits Protection against Salmonellosis in Mice

2004 ◽  
Vol 72 (7) ◽  
pp. 4297-4301 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
Jörg Hacker ◽  
Levente Emödy
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lethal Dose ◽  
Fold Increase ◽  
Oral Immunization ◽  
Oral Infection ◽  
Wild Type ◽  
Virulence Attenuation ◽  
Serovar Typhimurium

ABSTRACT Loss of the transcriptional antiterminator RfaH results in virulence attenuation (>104-fold increase in 50% lethal dose) of the archetypal Salmonella enterica serovar Typhimurium strain SL1344 by both orogastric and intraperitoneal routes of infection in BALB/c mice. Oral immunization with the mutant efficiently protects mice against a subsequent oral infection with the wild-type strain. Interestingly, in vitro immunoreactivity is not confined to strain SL1344; rather, it is directed also towards other serovars of S. enterica and even Salmonella bongori strains.

scholarly journals Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli

2002 ◽  
Vol 70 (8) ◽  
pp. 4406-4413 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
György Schneider ◽  
A. Salam Khan ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Mouse Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Serovar Typhimurium

ABSTRACT RfaH is a regulatory protein in Escherichia coli and Salmonella enterica serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of rfaH dramatically decreases the virulence of uropathogenic E. coli strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic rfaH mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its rfaH mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536rfaH::cat is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536rfaH::cat in comparison to the wild-type strain. trans complementation of the mutant strain with the rfaH gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection.

scholarly journals Host-Dependent Trigger of Caspases and Apoptosis by Legionella pneumophila

2007 ◽  
Vol 75 (6) ◽  
pp. 2903-2913 ◽  
Author(s):  
Marina Santic ◽  
Rexford Asare ◽  
Miljenko Doric ◽  
Yousef Abu Kwaik
Keyword(s):  
Single Cell ◽  
Legionella Pneumophila ◽  
Type Strain ◽  
Caspase 3 ◽  
Wild Type Strain ◽  
Wild Type ◽  
Human Macrophages ◽  
Caspase 1 ◽  
Mouse Macrophages ◽  
Late Stages

ABSTRACT The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.

scholarly journals Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 229-237 ◽  
Author(s):  
Arvind A. Bhagwat ◽  
Won Jun ◽  
Liu Liu ◽  
Porteen Kannan ◽  
Mahesh Dharne ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Gene Cassette ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Serovar Typhimurium ◽  
Resistance Gene Cassette ◽  
Reduced Rate

We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, opgG and opgH, form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The opgGH mutant strain was impaired in motility and growth under low osmolarity conditions. The opgGH mutation also resulted in a 2 log increase in the LD50 in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organism's lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the opgGH-defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of S. Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

scholarly journals Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division

2005 ◽  
Vol 187 (10) ◽  
pp. 3415-3420 ◽  
Author(s):  
Parampal Deol ◽  
Reena Vohra ◽  
Adesh Kumar Saini ◽  
Amit Singh ◽  
Harish Chandra ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Glucose Transport ◽  
Stress Responses ◽  
Type Strain ◽  
Wild Type Strain ◽  
Physiological Role ◽  
Wild Type ◽  
Bacterial Physiology ◽  
Transport Analysis

ABSTRACT Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of d-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

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