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1.
Clin Microbiol Infect ; 25(2): 252.e1-252.e4, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30195066

ABSTRACT

OBJECTIVES: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. METHODS: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. RESULTS: In HeLa and JEG-3 cells, the F2365 InlB+ strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB+ were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB+ strain (Mann-Whitney test). CONCLUSIONS: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.


Subject(s)
Bacterial Proteins/metabolism , Listeria monocytogenes/pathogenicity , Listeriosis/microbiology , Membrane Proteins/metabolism , Animals , Bacterial Proteins/genetics , Cell Line , Epidemics , Gene Expression Regulation, Bacterial , Humans , Listeria monocytogenes/genetics , Listeria monocytogenes/metabolism , Listeriosis/epidemiology , Liver/microbiology , Membrane Proteins/genetics , Mice , Point Mutation , Spleen/microbiology , Virulence
2.
J Invertebr Pathol ; 103(1): 24-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19800349

ABSTRACT

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry(-) with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.


Subject(s)
Host-Pathogen Interactions , Models, Biological , Moths/microbiology , Animals , Bacillus anthracis/metabolism , Bacillus anthracis/pathogenicity , Bacillus cereus/metabolism , Bacillus cereus/pathogenicity , Bacillus thuringiensis/metabolism , Bacillus thuringiensis/pathogenicity , Bacterial Toxins/metabolism , Enterococcus faecalis/metabolism , Enterococcus faecalis/pathogenicity , Larva/microbiology , Listeria monocytogenes/metabolism , Listeria monocytogenes/pathogenicity , Phylogeny , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Virulence
3.
Science ; 294(5543): 849-52, 2001 Oct 26.
Article in English | MEDLINE | ID: mdl-11679669

ABSTRACT

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.


Subject(s)
Bacterial Proteins/genetics , Genome, Bacterial , Listeria monocytogenes/genetics , Listeria/genetics , Adaptation, Physiological , Amino Acid Motifs , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Base Composition , Carrier Proteins/chemistry , Carrier Proteins/genetics , Chromosomes, Bacterial/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Transfer, Horizontal , Genes, Bacterial , Genomics , Listeria/chemistry , Listeria/physiology , Listeria monocytogenes/chemistry , Listeria monocytogenes/pathogenicity , Listeria monocytogenes/physiology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Sequence Analysis, DNA , Staphylococcus aureus/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Virulence/genetics
4.
Infect Immun ; 69(1): 529-33, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11119546

ABSTRACT

Superoxide dismutases (SODs) play an important role in protection against oxidative stress and have been shown to contribute to the pathogenicity of many bacterial species. To determine the function of the mycobacterial copper and zinc-cofactored SOD (CuZnSOD), we constructed and characterized Mycobacterium tuberculosis and Mycobacterium bovis BCG CuZnSOD null mutants. Both strains were more sensitive to superoxides and hydrogen peroxide than were their respective parental strains. The survival of M. bovis BCG in unstimulated as well as activated mouse bone marrow-derived macrophages was not affected by the loss of CuZnSOD. The survival of CuZnSOD deficient-M. tuberculosis in guinea pig tissues was comparable to that of its parental strain. These results indicate that the mycobacterial CuZnSOD is not essential for intracellular growth within macrophages and does not detectably contribute to the pathogenicity of M. tuberculosis.


Subject(s)
Mycobacterium tuberculosis/enzymology , Superoxide Dismutase/physiology , Animals , Bone Marrow Cells/microbiology , Female , Guinea Pigs , Hydrogen Peroxide/pharmacology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mycobacterium bovis/drug effects , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/growth & development , Uracil/metabolism
5.
J Bacteriol ; 181(11): 3402-8, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10348851

ABSTRACT

Exochelin is the primary extracellular siderophore of Mycobacterium smegmatis, and the iron-regulated fxbA gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557-569, 1994). We investigated the regulation of fxbA by the mycobacterial IdeR, a homolog of the Corynebacterium diphtheriae iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284-4289, 1995). Gel mobility shift experiments showed that IdeR binds to the fxbA regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the fxbA promoter that was identified in primer extension assays. fxbA regulation was measured in M. smegmatis wild-type and ideR mutant strains containing fxbA promoter-lacZ fusions. These experiments confirmed that fxbA expression is negatively regulated by iron and showed that inactivation of ideR results in iron-independent expression of fxbA. However, the levels of its expression in the ideR mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of fxbA.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Iron/pharmacology , Mycobacterium/genetics , Repressor Proteins , Transcription, Genetic/genetics , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Cations, Divalent/pharmacology , DNA Footprinting , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, Bacterial/genetics , Genes, Reporter , Hydroxymethyl and Formyl Transferases/genetics , Mutation , Mycobacterium/drug effects , Mycobacterium/enzymology , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , RNA, Messenger/analysis , RNA, Messenger/genetics , Response Elements/genetics , Sequence Homology, Amino Acid , Transcription, Genetic/drug effects , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
6.
Tuber Lung Dis ; 79(5): 287-98, 1999.
Article in English | MEDLINE | ID: mdl-10707257

ABSTRACT

SETTING: Low iron availability in the host induces the expression of iron acquisition systems and virulence genes in many pathogens. IdeR is a mycobacterial iron dependent regulator that controls the iron starvation and oxidative stress responses in Mycobacterium smegmatis. It is important to determine the role of IdeR and its regulon in M. tuberculosis, as identification of iron regulated genes can aid in the design of new drugs and generation of attenuated strains. OBJECTIVE: A potential IdeR binding site was found in the M. tuberculosis genome flanked by two divergently oriented open reading frames, irg1 and irg2. The aim of this study was to determine whether irg1 and irg2 were iron and IdeR regulated genes. DESIGN: Interaction of IdeR with the putative binding sequence was examined by gel shift and footprinting assays. Transcriptional fusions of irg1 and irg2 to IacZ were used to study the effect of iron levels on the expression of these genes. RESULTS: IdeR binds to the predicted binding site, which overlaps with the irg1 promoter. irg1 and irg2 expression was decreased by iron in M. tuberculosis and in wild type M. smegmatis, but not in a M. smegmatis ideR mutant. CONCLUSION: Two M. tuberculosis iron/IdeR regulated genes were identified. irg1 is predicted to be the M. tuberculosis hisE gene, which is involved in histidine biosynthesis. It is directly upstream of the M. tuberculosis hisG. irg2 encodes a putative membrane protein that is a member of the PPE family.


Subject(s)
Bacterial Proteins/genetics , Iron/physiology , Mycobacterium tuberculosis/genetics , Repressor Proteins , Binding Sites , DNA, Bacterial/genetics , Humans , Intracellular Signaling Peptides and Proteins , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/metabolism , Open Reading Frames , Polymerase Chain Reaction , Proteins/genetics , Regulatory Sequences, Nucleic Acid , Transcription, Genetic
7.
Trends Microbiol ; 6(9): 354-8, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9778728

ABSTRACT

Iron is an essential cofactor for vital functions in microorganisms. Bacterial pathogens have developed efficient iron-acquisition systems to counteract the defensive sequestration of iron by their hosts. In mycobacteria, the recently described protein, IdeR, negatively controls iron-uptake systems. This protein also has a role in the oxidative-stress response, as well as in resistance to the frontline antimycobacterial drug isoniazid.


Subject(s)
Antitubercular Agents/pharmacology , Iron/pharmacokinetics , Isoniazid/pharmacology , Mycobacterium/drug effects , Mycobacterium/physiology , Oxidative Stress/physiology , Antitubercular Agents/metabolism , Bacterial Proteins/metabolism , Drug Resistance, Microbial , Isoniazid/metabolism , Mycobacterium/genetics , Siderophores/physiology
9.
Tuber Lung Dis ; 79(2): 91-7, 1998.
Article in English | MEDLINE | ID: mdl-10645446

ABSTRACT

To understand how Mycobacterium tuberculosis survives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about sigma factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that sigma A is the essential housekeeping sigma factor and the alternative sigma factor sigma B, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in sigma A prevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatis and M. tuberculosis that are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosis gene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosis genes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosis grown in vitro with those found in bacteria growing in human macrophages.


Subject(s)
Gene Expression Regulation, Bacterial , Genes, Bacterial , Mycobacterium tuberculosis/genetics , Animals , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Humans , Iron/metabolism , Macrophages/microbiology , Mice , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Sigma Factor/genetics , Virulence/genetics
10.
Tuber Lung Dis ; 79(2): 99-106, 1998.
Article in English | MEDLINE | ID: mdl-10645447

ABSTRACT

OBJECTIVE: To understand the mechanism by which IdeR is necessary for maintaining wild type levels of KatG and SodA enzyme activity and normal isoniazid (INH) resistance. DESIGN: To identify the step(s) of SodA and KatG function that were affected by the ideR mutation, quantitative western immunoassays and ribonucleic acid (RNA) hybridizations were performed. To see if the increased INH sensitivity of the ideR mutant was caused by lower SodA activity, the Mycobacterium smegmatis sod gene was inactivated. RESULTS: The levels of KatG and SodA mRNA and protein in the M. smegmatis IdeR mutant are decreased to approximately 20-40% of those observed in the wild type parent strain. This is quantitatively similar to the decrease in KatG and SodA enzyme activities originally observed in the ideR strain. The M. smegmatis sodA mutant was slightly more sensitive to INH, compared to the wild type strain and was more resistant than the ideR mutant. CONCLUSION: IdeR is necessary for full expression of the M. smegmatis katG and sodA genes. It is not yet known whether this protein acts directly at the gene level. The lower levels of SodA contribute slightly to the increased susceptibility to INH of the ideR mutant, but cannot explain the magnitude of the INH sensitivity observed when IdeR is not present. These data suggest that IdeR is a regulator of the cellular stress response, as it has a protective role in cells facing environmental stresses, such as increased levels of reactive oxygen species and INH toxic intermediates. These conclusions do not necessarily apply to IdeR's role in M. tuberculosis physiology, since we have not inactivated its gene in this pathogen.


Subject(s)
Antitubercular Agents , Bacterial Proteins , Drug Resistance, Microbial/genetics , Genes, Bacterial , Isoniazid , Mycobacterium smegmatis/genetics , Reactive Oxygen Species , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Mutation , Mycobacterium smegmatis/enzymology , Peroxidases/analysis , Peroxiredoxins , Superoxide Dismutase/analysis
11.
Mol Microbiol ; 22(3): 535-44, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8939436

ABSTRACT

The mycobacterial ideR protein is a homologue of the diphtheria-toxin repressor DtxR. We have previously demonstrated that Mycobacterium tuberculosis ideR, like DtxR, represses transcription of Corynebacterium diphtheriae iron-regulated promoters in vivo and binds to C. diphtheriae operators in a metal-dependent manner in vitro. We show here that ideR mutants of M. smegmatis, constructed by allelic replacement, were defective in their ability to repress siderophore biosynthesis in the presence of iron. They were also more sensitive to hydrogen peroxide and had decreased levels of catalase/peroxidase (KatG) and manganese superoxide dismutase (Mn-SOD). This indicates that ideR is a negative regulator of siderophore production and is required for the response to superoxide- and hydrogen peroxide stress. We propose that ideR is the mycobacterial counterpart of the Escherichia coli Fur protein, i.e. It is a pleiotropic regulator that couples iron metabolism to the oxidative-stress response.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial , Mycobacterium/genetics , Oxidative Stress/genetics , Alleles , Blotting, Western , Catalase/biosynthesis , DNA Transposable Elements , DNA, Bacterial/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Ferric Compounds/chemical synthesis , Ferric Compounds/metabolism , Genetic Complementation Test , Hydrogen Peroxide/pharmacology , Iron/pharmacology , Mutagenesis, Insertional , Oxazoles/metabolism , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/chemical synthesis , Peroxidase/biosynthesis , Plasmids , Recombination, Genetic , Repressor Proteins/genetics , Repressor Proteins/physiology , Siderophores/metabolism , Superoxide Dismutase/biosynthesis , Transformation, Genetic
12.
Gene ; 165(1): 67-70, 1995 Nov 07.
Article in English | MEDLINE | ID: mdl-7489918

ABSTRACT

We have previously described sigma A and sigma B and their structural genes, mysA and mysB, respectively, in Mycobacterium smegmatis. We have now sequenced the corresponding regions in the M. tuberculosis and M. leprae chromosomes, and have found the two homologous genes. The chromosomal linkage and the deduced amino acid (aa) sequences of the two genes show very high similarity in the three species of mycobacteria. We also report the finding of two other open reading frames (ORF) in these clusters. orfX, which has an unknown function, is located between mysA and mysB. The other ORF, located downstream from mysB, encodes a homolog of DtxR, the iron regulatory protein from Corynebacterium diphtheriae (Cd).


Subject(s)
Genome, Bacterial , Mycobacterium leprae/genetics , Mycobacterium tuberculosis/genetics , Sigma Factor/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Molecular Sequence Data , Multigene Family , Sequence Alignment
14.
Appl Environ Microbiol ; 60(3): 953-9, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8161186

ABSTRACT

A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that are major cell culture contaminants belonging to the class Mollicutes were investigated: Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma fermentans. After a concentration step involving seeded sera, genus-specific primers were used to amplify a 717-bp DNA fragment within the 16S rRNA gene of mycoplasmas. In a second step, the universal PCR was followed by amplification of variable regions of the 16S rRNA gene by using species-specific primers, which allowed identification of contaminant mycoplasmas. With this method, 10 fg of purified DNA and 1 to 10 color-changing units of mycoplasmas could be detected. Since the sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal mycoplasma-specific 32P-labelled oligonucleotide probe, a detection limit of 1 to 10 genome copies per PCR sample was obtained. This highly sensitive, specific, and simple assay may be a useful alternative to methods currently used to detect mycoplasmas in animal sera.


Subject(s)
Polymerase Chain Reaction/methods , Tenericutes/isolation & purification , Animals , Base Sequence , Blood Specimen Collection , Cattle , Horses , Molecular Sequence Data , Sensitivity and Specificity , Species Specificity , Time Factors
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