PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.     

Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency

The Bacillus subtilis PerR repressor regulates the adaptive response to peroxide stress. The PerR regulon includes the major vegetative catalase (katA), an iron storage protein (mrgA), an alkylhydroperoxide reductase (ahpCF), a zinc uptake system (zosA), heme biosynthesis enzymes (hemAXCDBL), the iron uptake repressor (fur), and perR itself. A perR null strain is resistant to hydrogen peroxide, accumulates a porphyrin-like compound, and grows very slowly. The poor growth of the perR mutant can be largely accounted for by the elevated expression of two proteins: the KatA catalase and Fur. Genetic studies support a model in which poor growth of the perR null mutant is due to elevated repression of iron uptake by Fur, exacerbated by heme sequestration by the abundant catalase protein. Analysis of the altered-function allele perR991 further supports a link between PerR and iron homeostasis. Strains containing perR991 are peroxide resistant but grow nearly as well as the wild type. Unlike a perR null allele, the perR991 allele (F51S) derepresses KatA, but not Fur, which likely accounts for its comparatively rapid growth.     

Cloning and characterization of two novel DNases from Streptococcus pyogenes

The proteins in the culture supernatant (exoproteins) from Streptococcus pyogenes serotype M1 were separated by two-dimensional gel electrophoresis, and their N-terminal amino acid sequences were determined. The amino acid sequences were compared to sequences in the S. pyogenes genome database. The coding sequence showed similarity to sequences of two genes, mf2-v ( mf2 variant) and mf3, which had sequence similarity to genes encoding mitogenic factor (MF); MF has DNase activity. The recombinant genes were expressed in Escherichia coli and the proteins were synthesized. Mf2-v and Mf3 had DNase activity. The activity of Mf2-v was localized to the C-terminal half of the protein. The mf3 gene was shown to be present in most clinically isolated strains of S. pyogenes tested, and the mf2gene was detected in 20% of the isolates. The products of the mf2 and mf3 genes in clinically isolated S. pyogenes strains were thus shown to be DNases.     

Using the Ralstonia solanacearum Tat secretome to identify bacterial wilt virulence factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself.     

Generation of a mature streptococcal cysteine proteinase is dependent on cell wall-anchored M1 protein

In the present study, we have generated a mutant strain of Streptococcus pyogenes, MC25, which lacks M protein on its surface, and we demonstrate that this strain is unable to generate a mature 28 kDa cysteine proteinase. Furthermore, we show that S. pyogenes bacteria of M1 serotype are dependent on cell wall-anchored M protein to cleave the secreted zymogen into a mature cysteine proteinase. We also show that MC25 secretes a 40 kDa zymogen, having a conformation different from that secreted by wild-type bacteria. We provide data showing that the cleavage site is not blocked but, presumably, the active site is. This suggests that M protein, when anchored to the cell wall, is involved in the unfolding of the zymogen and generation of a mature cysteine proteinase that can be activated under reducing conditions. Our data add new aspects to the interaction between two important virulence factors of S. pyogenes, the streptococcal cysteine proteinase and M protein.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.     

Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.