- Volume 156, Issue 12, 2010
Volume 156, Issue 12, 2010
- Lecture
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Microbial communication and virulence: lessons from evolutionary theory
More LessAt the heart of tackling the huge challenge posed by infectious micro-organisms is the overwhelming need to understand their nature. A major question is, why do some species of bacteria rapidly kill their host whilst others are relatively benign? For example, Yersinia pestis, the causative organism of plague, is a highly virulent human pathogen whilst the closely related Yersinia pseudotuberculosis causes a much less severe disease. Using molecular techniques such as mutating certain genes, microbiologists have made significant advances over recent decades in elucidating the mechanisms that govern the production of virulence factors involved in causing disease in many bacterial species. There are also evolutionary and ecological factors which will influence virulence. Many of these ideas have arisen through the development of evolutionary theory and yet there is strikingly little empirical evidence testing them. By applying both mechanistic and adaptive approaches to microbial behaviours we can begin to address questions such as, what factors influence cooperation and the evolution of virulence in microbes and can we exploit these factors to develop new antimicrobial strategies?
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- Cell And Molecular Biology Of Microbes
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Transcriptional autoregulation of the RcsCDB phosphorelay system in Salmonella enterica serovar Typhimurium
More LessThe RcsCDB (Rcs) phosphorelay system is involved in the regulation of many envelope genes, such as those responsible for capsule synthesis, flagella production and O-antigen chain length, as well as in other cellular activities of several enteric bacteria. The system is composed of three proteins: the sensor RcsC, the response regulator RcsB, and the phospho-transfer intermediary protein RcsD. Previously, we reported two important aspects of this system: (a) rcsB gene expression is under the control of P rcsDB and P rcsB promoters, and (b) rcsD gene transcription decreases when the bacteria reach high levels of the RcsB regulator. In the present work, we demonstrate that the RcsB protein represses rcsD gene expression by binding directly to the P rcsDB promoter, negatively autoregulating the Rcs system. Furthermore, we report the physiological role of the RcsB regulator, which is able to modify bacterial swarming behaviour when expressed under the control of the P rcsB promoter.
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Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans
Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.
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Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition
More LessBacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β′ subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β′ and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β′ and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β′. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.
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DevT (Alr4674), resembling a Ser/Thr protein phosphatase, is essential for heterocyst function in the cyanobacterium Anabaena sp. PCC 7120
Heterocyst-forming cyanobacteria are able to perform oxygenic photosynthesis and nitrogen fixation simultaneously in the same filament, by restricting the highly O2-sensitive nitrogenase to specialized cells, the heterocysts. A remarkable change in morphology and metabolism accompanies the differentiation of heterocysts, which only occurs when no source of combined nitrogen is available. In this study, we characterized DevT (Alr4674), a putative protein phosphatase from Anabaena PCC 7120. Mutants defective in devT are able to form morphologically mature heterocysts, which however cannot fix N2, and the mutant cannot survive without a source of combined nitrogen. DevT shows homology to phosphatases of the PPP family and displays a Mn2+-dependent phosphatase activity that can be inhibited by phosphatase inhibitors and oxidizing conditions. DevT is constitutively expressed in both vegetative cells and heterocysts, and is not regulated by NtcA. The heterocyst regulator HetR may exert a certain inhibition on the expression of devT. Under diazotrophic growth conditions, DevT protein accumulates specifically in mature heterocysts. Therefore DevT plays a still unknown role in a late essential step of heterocyst differentiation.
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Peroxynitrite stress is exacerbated by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide
More LessOxidative and nitrosative stresses including nitric oxide (NO), superoxide () and peroxynitrite play key roles in determining the outcome of bacterial infections. In order to survive within the host and allow proliferation within immune cells such as macrophages, Salmonella isolates have a number of inducible proteins that are able to detoxify these highly reactive species, notably the anoxically functioning NO reductase NorVW, and the aerobically functioning flavohaemoglobin, Hmp, which catalyses the reaction between oxygen and NO to produce relatively inert nitrate. However, in the absence of NO but in the presence of reducing substrates and oxygen, is generated from Hmp-mediated electron transfer to bound oxygen and may form a variety of further oxidative species. Hence, Hmp expression is under tight negative regulation by the transcription factor NsrR, abolition of which causes an increase in the production of Hmp. In a previous study, this increase in Hmp levels conferred resistance to the nitrosating agent S-nitrosoglutathione but, perhaps surprisingly, the organism became more sensitive to killing by macrophages. Here, we report that an nsrR mutant that constitutively overexpresses Hmp is also hypersensitive to peroxynitrite in vitro. This sensitivity is alleviated by deletion of the hmp gene or pre-incubation of growing bacteria with NO-releasing agents. We hypothesize that Hmp-expressing cells, in the absence of NO, generate reactive oxygen species, the toxicity of which is exacerbated by peroxynitrite in vitro and in macrophages. RT-PCR confirmed that peroxynitrite causes oxidative stress and upregulation of katG and ahpC, whilst hmp and norV expression are affected very little. The katG gene upregulated by peroxynitrite encodes a catalase peroxidase enzyme with well-established roles in detoxifying peroxides. Here, we report that KatG is also able to enhance the breakdown of peroxynitrite, suggesting that the protective role of this enzyme may be wider than previously thought. These data suggest that spatial and temporal fluctuations in the levels of NO and reactive oxygen species will have important consequences for bacterial survival in the macrophage.
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Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus Clonostachys rosea in complex with the inhibitor caffeine
Chitinases are a group of enzymes capable of hydrolysing the β-(1,4)-glycosidic bonds of chitin, an essential component of the fungal cell wall, the shells of nematode eggs, and arthropod exoskeletons. Chitinases from pathogenic fungi have been shown to be putative virulence factors, and can play important roles in infecting hosts. However, very limited information is available on the structure of chitinases from nematophagous fungi. Here, we present the 1.8 Å resolution of the first structure of a Family 18 chitinase from this group of fungi, that of Clonostachys rosea CrChi1, and the 1.6 Å resolution of CrChi1 in complex with a potent inhibitor, caffeine. Like other Family 18 chitinases, CrChi1 has the DXDXE motif at the end of strand β5, with Glu174 as the catalytic residue in the middle of the open end of the (β/α)8 barrel. Two caffeine molecules were shown to bind to CrChi1 in subsites −1 to +1 in the substrate-binding domain. Moreover, site-directed mutagenesis of the amino acid residues forming hydrogen bonds with caffeine molecules suggests that these residues are important for substrate binding and the hydrolytic process. Our results provide a foundation for elucidating the catalytic mechanism of chitinases from nematophagous fungi and for improving the pathogenicity of nematophagous fungi against agricultural pest hosts.
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A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal
More LessBacillus thuringiensis is the leading biopesticide used to control insect pests worldwide. Although they have a long record of safe use, under certain conditions commercial strains of B. thuringiensis have the ability to produce numerous putative enterotoxins that have been associated with food poisoning attributed to Bacillus cereus. Therefore, we designed a strategy to delete the genes encoding these toxins. B. thuringiensis strain VBTS 2477 contained genes encoding NHE, CytK-2 and three homologues of haemolysin BL (HBL, HBLa1 and HBLa2). This is the first report, to our knowledge, of a strain of B. cereus or B. thuringiensis containing three sets of hbl operons. The genes encoding HBLa1 and HBLa2 were 96–97 % identical to each other and 76–84 % identical to those encoding HBL. The hbla2 operon was detected by PCR amplification only after hbla1 was deleted. We used sequential gene replacement to replace the wild-type copies of the NHE and three HBL operons with copies that contained internal deletions that span the three genes in each operon. The insecticidal activity of the quadruple-enterotoxin-deficient mutant was similar to that of the wild-type strain against larvae of Trichoplusia ni, Spodoptera exigua and Plutella xylostella. This demonstrates that the genes for enterotoxins can be deleted, eliminating the possibility of enterotoxin production without compromising the insecticidal efficacy of a strain of B. thuringiensis.
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Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane
Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 μm. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported.
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- Environmental And Evolutionary Microbiology
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Variation in the Neisseria meningitidis FadL-like protein: an evolutionary model for a relatively low-abundance surface antigen
More LessThe molecular diversity of a novel Neisseria meningitidis antigen, encoded by the ORF NMB0088 of MC58 (FadL-like protein), was assessed in a panel of 64 diverse meningococcal strains. The panel consisted of strains belonging to different serogroups, serotypes, serosubtypes and MLST sequence types, of different clinical sources, years and countries of isolation. Based on the sequence variability of the protein, the FadL-like protein has been divided into four variant groups in this species. Antigen variants were associated with specific serogroups and MLST clonal complexes. Maximum-likelihood analyses were used to determine the relationships among sequences and to compare the selection pressures acting on the encoded protein. Furthermore, a model of population genetics and molecular evolution was used to detect natural selection in DNA sequences using the non-synonymous : synonymous substitution (d N : d S) ratio. The meningococcal sequences were also compared with those of the related surface protein in non-pathogenic commensal Neisseria species to investigate potential horizontal gene transfer. The N. meningitidis fadL gene was subject to only weak positive selection pressure and was less diverse than meningococcal major outer-membrane proteins. The majority of the variability in fadL was due to recombination among existing alleles from the same or related species that resulted in a discrete mosaic structure in the meningococcal population. In general, the population structuring observed based on the FadL-like membrane protein indicates that it is under intermediate immune selection. However, the emergence of a new subvariant within the hyperinvasive lineages demonstrates the phenotypic adaptability of N. meningitidis, probably in response to selective pressure.
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- Genes And Genomes
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Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum
More LesspCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem–loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.
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ccrAB Ent serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium
The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrAB Ent genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrAB Ent genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrA Ent probe (n=76) and partial DNA sequencing of ccrA Ent and ccrB Ent genes (n=38). ccrAB Ent genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrAB Ent genes were not found. Thirty-eight sequenced ccrAB Ent genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrAB Ent flanking chromosomal genes. Expression analysis of ccrAB Ent genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrAB Ent mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrAB Ent genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrAB Ent positive and negative isolates, suggesting acquisition or loss of ccrAB Ent in E. faecium. In summary, ccrAB Ent genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups.
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- Microbial Pathogenicity
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Candida albicans forms biofilms on the vaginal mucosa
More LessCurrent understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).
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Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody
More LessDespite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo.
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Streptococcal inhibitor of complement-mediated lysis (SIC): an anti-inflammatory virulence determinant
Since the late 1980s, a worldwide increase of severe Streptococcus pyogenes infections has been associated with strains of the M1 serotype, strains which all secrete the streptococcal inhibitor of complement-mediated lysis (SIC). Previous work has shown that SIC blocks complement-mediated haemolysis, inhibits the activity of antibacterial peptides and has affinity for the human plasma proteins clusterin and histidine-rich glycoprotein; the latter is a member of the cystatin protein family. The present work demonstrates that SIC binds to cystatin C, high-molecular-mass kininogen (HK) and low-molecular-mass kininogen, which are additional members of this protein family. The binding sites in HK are located in the cystatin-like domain D3 and the endothelial cell-binding domain D5. Immobilization of HK to cellular structures plays a central role in activation of the human contact system. SIC was found to inhibit the binding of HK to endothelial cells, and to reduce contact activation as measured by prolonged blood clotting time and impaired release of bradykinin. These results suggest that SIC modifies host defence systems, which may contribute to the virulence of S. pyogenes strains of the M1 serotype.
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Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis
More LessPathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α-l-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and d-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4–6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.
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Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells
More LessRecently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.
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Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2
More LessFor successful infection, Salmonella enterica secretes and injects effector proteins into host cells by two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands (SPIs)-1 and -2. The SPI-2 T3SS is involved in intracellular survival of S. enterica serovar Typhimurium and systemic disease. As little is known regarding the function of the SPI-2 T3SS from S. enterica serovar Typhi, the aetiological agent of typhoid fever, we investigated its role for survival in human macrophages. Mutations in the translocon (sseB), basal secretion apparatus (ssaR) and regulator (ssrB) did not result in any reduction in survival under many of the conditions tested. Similar results were obtained with another S. Typhi strain or by using human primary cells. Results were corroborated based on complete deletion of the SPI-2 T3SS. Surprisingly, the data suggest that the SPI-2 T3SS of S. Typhi is not required for survival in human macrophages.
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Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS− strains generate small colony variants in vivo that are distinct from in vitro variants
Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS− strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS− did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS− parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.
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Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target
More LessStaphylococcus aureus is a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence of S. aureus to human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437–464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis with S. aureus strain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B–CK8 interaction is a novel factor in S. aureus infections.
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Role of GacA in virulence of Vibrio vulnificus
The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.
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- Physiology And Biochemistry
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Arginine metabolism in Trichomonas vaginalis infected with Mycoplasma hominis
Both Mycoplasma hominis and Trichomonas vaginalis utilize arginine as an energy source via the arginine dihydrolase (ADH) pathway. It has been previously demonstrated that M. hominis forms a stable intracellular relationship with T. vaginalis; hence, in this study we examined the interaction of two localized ADH pathways by comparing T. vaginalis strain SS22 with the laboratory-generated T. vaginalis strain SS22-MOZ2 infected with M. hominis MOZ2. The presence of M. hominis resulted in an approximately 16-fold increase in intracellular ornithine and a threefold increase in putrescine, compared with control T. vaginalis cultures. No change in the activity of enzymes of the ADH pathway could be demonstrated in SS22-MOZ2 compared with the parent SS22, and the increased production of ornithine could be attributed to the presence of M. hominis. Using metabolic flow analysis it was determined that the elasticity of enzymes of the ADH pathway in SS22-MOZ2 was unchanged compared with the parent SS22; however, the elasticity of ornithine decarboxylase (ODC) in SS22 was small, and it was doubled in SS22-MOZ2 cells. The potential benefit of this relationship to both T. vaginalis and M. hominis is discussed.
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Structural studies of cord factors from Mycobacterium simiae related to the capacity for tumour necrosis factor alpha (α-TNF) induction
More LessThe structure of cord factor was studied in several strains of Mycobacterium simiae, including ‘habana’ TMC 5135, considered as highly immunogenic in experimental tuberculosis and leprosy. The mycolic acids liberated from cord factor were identified in all cases as α′-, α- and keto-mycolates. According to the general NMR and MS data, α′-mycolates were mono-unsaturated and contained from 64 to 68 carbon atoms, whereas α-mycolates mainly presented two 2,3-disubstituted cyclopropane rings and a chain length of 80–91 carbon atoms; keto-mycolates mostly contained one cyclopropane ring and 85–91 carbon atoms. Taking into account the 1H-NMR results, strains varied in the ratio of the different mycolates, and the high levels of keto-mycolates found in the cord factors of TMC 5135 and ATCC 25275T stood out. Notably, MS revealed that the odd carbon number series of α-mycolates (C87–C89) predominated in the cord factor of TMC 5135, in contrast to the remaining studied strains, in which the even (C84–C86) and odd carbon number series appeared more equal. The fine structural differences detected among the cord factors studied did not seem to be relevant to the general capacity of these molecules to induce the secretion of tumour necrosis factor alpha, as the cord factors from several strains of M. simiae (TMC 5135, IPK-342 and ATCC 25275T) induced similar amounts of this cytokine in RAW 264.7 cells.
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FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter
Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H+ symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K m 0.24±0.04 mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.
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Phenotypic and physiological alterations by heterologous acylhomoserine lactone synthase expression in Pseudomonas putida
More LessMany bacteria harbour an incomplete quorum-sensing (QS) system, whereby they possess LuxR homologues without the QS acylhomoserine lactone (AHL) synthase, which is encoded by a luxI homologue. An artificial AHL-producing plasmid was constructed using a cviI gene encoding the C6-AHL [N-hexanoyl homoserine lactone (HHL)] synthase from Chromobacterium violaceum, and was introduced successfully into both the wild-type and a ppoR (luxR homologue) mutant of Pseudomonas putida. Our data provide evidence to suggest that the PpoR–HHL complex, but neither PpoR nor HHL alone, could attenuate growth, antibiotic resistance and biofilm formation ability. In contrast, swimming motility, siderophore production and indole degradation were enhanced by PpoR–HHL. The addition of exogenous indole increased biofilm formation and reduced swimming motility. Interestingly, indole proved ineffective in the presence of PpoR–HHL, thereby suggesting that the PpoR–HHL complex masks the effects of indole. Our data were supported by transcriptome analyses, which showed that the presence of the plasmid-encoded AHL synthase altered the expression of many genes on the chromosome in strain KT2440. Our results showed that heterologous luxI expression that occurs via horizontal gene transfer can regulate a broad range of specific target genes, resulting in alterations of the phenotype and physiology of host cells.
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Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases
More LessBacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (NrfI, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as CX2CH (by CcsA2), CX2CK (by NrfI) and CX15CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of NrfI and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs.
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HrcA and DnaK are important for static and continuous-flow biofilm formation and disinfectant resistance in Listeria monocytogenes
More LessThe food-borne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Since biofilms are generally difficult to eradicate during clean-up procedures, they pose a major risk for the food industry. Stress resistance mechanisms involved in L. monocytogenes biofilm formation and disinfectant resistance have, to our knowledge, not been identified thus far. In this study, we investigated the role of hrcA, which encodes the transcriptional regulator of the class I heat-shock response, and dnaK, which encodes a class I heat-shock response chaperone protein, in static and continuous-flow biofilm formation and resistance against benzalkonium chloride and peracetic acid. Induction of both hrcA and dnaK during continuous-flow biofilm formation was observed using quantitative real-time PCR and promoter reporters. Furthermore, in-frame deletion and complementation mutants of hrcA and dnaK revealed that HrcA and DnaK are required to reach wild-type levels of both static and continuous-flow biofilms. Finally, disinfection treatments of planktonic-grown cells and suspended static and continuous-flow biofilm cells of wild-type and mutants showed that HrcA and DnaK are important for resistance against benzalkonium chloride and peracetic acid. In conclusion, our study revealed that HrcA and DnaK are important for L. monocytogenes biofilm formation and disinfectant resistance.
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6S RNA regulation of relA alters ppGpp levels in early stationary phase
More Less6S RNA is a small, non-coding RNA that interacts directly with σ 70-RNA polymerase and regulates transcription at many σ 70-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ 70-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σ S activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions.
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Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus
More LessAn extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA–GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA–GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA–GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a hetero-oligomeric GDH that can be regulated allosterically.
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Volatile organic compound production by organisms in the genus Ascocoryne and a re-evaluation of myco-diesel production by NRRL 50072
More LessThe Patagonian fungal endophyte NRRL 50072 is reported to produce a variety of medium-chain and highly branched volatile organic compounds (VOCs) that have been highlighted for their potential as fuel alternatives and are collectively termed myco-diesel. To assess the novelty of this observation, we determined the extent to which ten closely related Ascocoryne strains from commercial culture collections possess similar VOC production capability. DNA sequencing established a high genetic similarity between NRRL 50072 and each Ascocoryne isolate, consistent with its reassignment as Ascocoryne sarcoides. The Ascocoryne strains did not produce highly branched medium-chain-length alkanes, and efforts to reproduce the branched alkane production of NRRL 50072 were unsuccessful. However, we confirmed the production of 30 other products and expanded the list of VOCs for NRRL 50072 and members of the genus Ascocoryne. VOCs detected from the cultures consisted of short- and medium-chain alkenes, ketones, esters and alcohols and several sesquiterpenes. Ascocoryne strains NRRL 50072 and CBS 309.71 produced a more diverse range of volatiles than the other isolates tested. CBS 309.71 also showed enhanced production compared with other strains when grown on cellulose agar. Collectively, the members of the genus Ascocoryne demonstrated production of over 100 individual compounds, with a third of the short- and medium-chain compounds also produced when cultures were grown on a cellulose substrate. This comparative production analysis could facilitate future studies to identify and manipulate the biosynthetic machinery responsible for production of individual VOCs, including several that have a potential application as biofuels.
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Volumes and issues
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Volume 170 (2024)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)