BMC Microbiology - Latest articles

Background: The HIV pandemic raised the potential for facultative-pathogenic mycobacterial species like, Mycobacterium kansasii, to cause disseminating disease in humans with immune deficiencies. In contrast, non-pathogenic mycobacterial species, like M. smegmatis, are not known to cause disseminating disease even in immunocompromised individuals. We hypothesized that this difference in phenotype could be explained by the strong induction of an innate immune response by the non-pathogenic mycobacterial species. Results: A comparison of two rapid-growing, non-pathogenic species (M. smegmatis and M. fortuitum) with two facultative-pathogenic species (M. kansasii and M. bovis BCG) demonstrated that only the non-pathogenic bacteria induced strong apoptosis in human THP-1 cells and murine bone marrow-derived macrophages (BMDM) and dendritic cells (BMDD). The phospho-myo-inositol modification of lipoarabinomannan (PI-LAM) isolated from non-pathogenic species may be one of the cell wall components responsible for the pro-inflammatory activity of the whole bacteria. Indeed, PI-LAM induces high levels of apoptosis and IL-12 expression compared to the mannosyl modification of LAM isolated from facultative-pathogenic mycobacteria. The apoptosis induced by non-pathogenic M. smegmatis was dependent upon caspase-3 activation and TNF secretion. Consistently, BALB/c BMDM responded by secreting large amounts of TNF upon infection with non-pathogenic but not facultative-pathogenic mycobacteria. Interestingly, C57Bl/6 BMDM do not undergo apoptosis upon infection with non-pathogenic mycobacteria despite the fact that they still induce an increase in TNF secretion. This suggests that the host cell signaling pathways are different between these two mouse genotypes and that TNF is necessary but not sufficient to induce host cell apoptosis. Conclusion: These results demonstrate a much stronger induction of the innate immune response by non-pathogenic versus facultative-pathogenic mycobacteria as measured by host cell apoptosis, IL-12 and TNF cytokine induction. These observations lend support to the hypothesis that the strong induction of the innate immune response is a major reason for the lack of pathogenicity in fast-growing mycobacteria.

Background: Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group. Results: Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDA adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. Conclusions: We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

Background: Leucine rich repeats (LRRsare present in over 60,000 proteins that have been identified in viruses, bacteria, archae, and eukaryotes. All known structures of repeated LRRs adopt an arc shape. Most LRRs are 20-30 residues long. All LRRs contain LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. Seven classes of LRRs have been identified. However, other LRR classes remains to be characterized. The evolution of LRRs is not well understood. Results: Here we describe a novel LRR domain, or nested repeat observed in 134 proteins from 54 bacterial species. This novel LRR domain has 21 residues with the consensus sequence of LxxLxLxxNxLxxLDLxx(N/L/Q/x)xx or LxxLxCxxNxLxxLDLxx(N/L/x)xx. This LRR domain is characterized by a nested periodicity; it consists of alternating 10- and 11- residues units of LxxLxLxxNx(x/-). We call it "IRREKO" LRR, since the Japanese word for "nested" is "IRREKO". The first unit of the "IRREKO" LRR domain is frequently occupied by an "SDS22-like" LRR with the consensus of LxxLxLxxNxLxxLxxLxxLxx or a "Bacterial" LRR with the consensus of LxxLxLxxNxLxxLPxLPxx. In some proteins an "SDS22-like" LRR intervenes between "IRREKO" LRRs.Conclusion Proteins having "IRREKO" LRR domain are almost exclusively found in bacteria. It is suggested that IRREKO@LRR evolved from a common ancestor with "SDS22-like" and "Bacterial" classes and that the ancestor of IRREKO@LRR is 10 or 11 residues of LxxLxLxxNx(x/-). The "IRREKO" LRR is predicted to adopt an arc shape with smaller curvature in which beta-strands are formed on both concave and convex surfaces.

Background: Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail. Results: We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (cysK-cysE, cysP1 and cysP2) or an S-box riboswitch (metK and metT). We also showed that the ubiG operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA.In addition, we found that expression of pfoA (theta-toxin), nagL (one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation. Conclusion: Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation.

Background: The aim of the present study was to assess possible health effects of airway exposures to Bacillus thuringiensis (Bt) based biopesticides in mice. Endpoints were lung inflammation, clearance of bacteria from the lung lumen and histological alteration. Hazard identifications of the biopesticides were carried out using intratracheal (i.t.) instillation, followed by an inhalation study.The two commercial biopesticides used were based on the Bt. subspecies kurstaki and israelensis, respectively. Groups of BALB/c mice were i.t instilled with one bolus (3.5x105 or 3.4x106 colony forming units (CFU) per mouse) of either biopesticide. Control mice were instilled with sterile water. Bronchoalveolar lavage fluid (BALF) were collected and the inflammatory cells in the BALF were counted and differentiated. The BALF were also subjected to CFU counts. Results: BALF cytology showed an acute inflammatory response dominated by neutrophils 24 hours after instillation of biopesticide. Four days after instillation, the neutrophil number was normalised and inflammation was dominated by lymphocytes and eosinophils, whereas 70 days after instillation, the inflammation was interstitially located with few inflammatory cells present in the lung lumen.Half of the instilled mice had remaining CFU recovered from BALF 70 days after exposure.To gain further knowledge with relevance for risk assessment, mice were exposed to aerosols of biopesticide one hour per day for 2x5 days. Each mouse received 1.9x10^4 CFU Bt israelensis or 2.3x10^3 CFU Bt kurstaki per exposure. Seventy days after end of the aerosol exposures, 3 out of 17 mice had interstitial lung inflammation. CFU could be recovered from 1 out of 10 mice 70 days after exposure to aerosolised Bt kurstaki. Plethysmography showed that inhalation of Bt aerosol did not induce airway irritation. Conclusions: Repeated low dose aerosol exposures to commercial Bt based biopesticides can induce sub-chronic lung inflammation in mice, which may be the first step in the development of chronic lung diseases. Inhalation of Bt aerosols does not induce airway irritation, which could explain why workers may be less inclined to use a filter mask during the application process, and are thereby less protected from exposure to Bt spores.

Background: Poultry meat is one of the most important sources of human campylobacteriosis, an acute bacterial enteritis which is a major problem worldwide. Campylobacter coli and Campylobacter jejuni are the most common Campylobacter species associated with this disease. These pathogens live in the intestinal tract of most avian species and under commercial conditions they spread rapidly to infect a high proportion of the flock, which makes their treatment and prevention very difficult. Bacteriophages (phages) are naturally occurring predators of bacteria with high specificity and also the capacity to evolve to overcome bacterial resistance. Therefore phage therapy is a promising alternative to antibiotics in animal production. This study tested the efficacy of a phage cocktail composed of three phages for the control of poultry infected with C.coli and C.jejuni. Moreover, it evaluated the effectiveness of two routes of phage administration (by oral gavage and in feed) in order to provide additional information regarding their future use in a poultry unit. Results: The results indicate that experimental colonisation of chicks was successful and that the birds showed no signs of disease even at the highest dose of Campylobacter administered. The phage cocktail was able to reduce the titre of both C.coli and C.jejuni in faeces by approximately 2log10cfu/g when administered by oral gavage and in feed. This reduction persisted throughout the experimental period and neither pathogen regained their former numbers. The reduction in Campylobacter titre was achieved earlier (2 days post-phage administration) when the phage cocktail was incorporated in the birds' feed. Campylobacter strains resistant to phage infection were recovered from phage-treated chickens at a frequency of 13%. These resistant phenotypes did not exhibit a reduced ability to colonize the chicken guts and did not revert to sensitive types. Conclusions: Our findings provide further evidence of the efficacy of phage therapy for the control of Campylobacter in poultry. The broad host range of the novel phage cocktail enabled it to target both C.jejuni and C.coli strains. Moreover the reduction of Campylobacter by approximately 2 log10cfu/g, as occurred in our study, could lead to a 30-fold reduction in the incidence of campylobacteriosis associated with consumption of chicken meals (according to mathematical models). To our knowledge this is the first report of phage being administered in feed to Campylobacter-infected chicks and our results show that it lead to an earlier and more sustainable reduction of Campylobacter than administration by oral gavage. Therefore the present study is of extreme importance as it has shown that administering phages to poultry via the food could be successful on a commercial scale.

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma54-dependent manner. A more complete picture of the sigma54 regulon was achieved by combining the transcriptome data with an in silico search for potential sigma54-dependent promoters, using a position weight matrix approach. One of these sigma54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma54-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma54 regulon.

Background: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, beta-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

Background: Acidithiobacillus ferrooxidans is chemolithoautotrophic gamma-proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO2 uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study. Results: Four gene clusters (termed cbb1-4) in the A. ferrooxidans genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson-Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO2-concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon cbb1 is divergently transcribed from a gene, cbbR, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma70-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N11-A/TNA-N7TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the cbb1, cbb2 and cbb3 operons, demonstrating that the predicted CbbR-binding sites are functional in vitro. However, CbbR failed to bind the upstream region of the cbb4 operon that contains cbbP, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The cbb3 operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO2 fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of A. ferrooxidans to fix CO2 at different concentrations of CO2. Conclusions: A. ferrooxidans has features of cbb gene organization for CO2-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the cbbP gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation.

Background: Salmonella enterica is a facultative intracellular pathogen that replicates within a membrane-bound compartment termed Salmonella containing vacuole (SCV). The biogenesis of SCV requires Salmonella type III protein secretion/translocation system and their effector proteins which are translocated into host cells to exploit the vesicle trafficking pathways. SseF is one of these effectors required for SCV formation and Intracellular Salmonella replication through unknown mechanisms. Results: In an attempt to identify host proteins that interact with SseF, we conduct a yeast two-hybrid screening of human cell cDNA library using SseF as the bait. We identified that TIP60, an acetyltransferase, interacts with SseF. We showed that the TIP60 acetylation activity was increased in the presence of SseF, and TIP60 was upregulated upon Salmonella infection. In addition, TIP60 is required for efficient intracellular Salmonella replication in macrophages. Conclusion: Taken together, our data suggest that Salmonella may use SseF to exploit the host TIP60 acetyltransferase activity to promote efficient Salmonella replication inside host cells.