- Volume 157, Issue 6, 2011

Campylobacter jejuni lacks the enzyme phosphofructokinase and, consequently, is incapable of metabolizing glucose. Instead, the pathogen uses a number of other chemicals to serve as electron donors. Like chemolithotrophic bacteria, C. jejuni is able to respire sulphite in the presence of a sulphite : cytochrome c oxidoreductase (SOR) that is encoded by the genes cj0004c and cj0005c; the former encodes a monohaem cytochrome c oxidoreductase and the latter a molybdopterin oxidoreductase. After screening of a transposon-based mutant library, we identified a mutant with an insertion in gene cj0005c that was strongly reduced in its capacity to infect Caco2 cells. Further characterization of a corresponding non-random knockout mutant together with a complemented mutant and the parental strain showed the cj0005c-deficient mutant to exhibit clearly reduced motility and diminished adherence to host cells. Furthermore, the transcription of genes responsible for the synthesis of, in particular, legionaminic acid was downregulated and the mutant had a reduced capacity to autoagglutinate. In contrast, neither the proliferation of the mutant, nor its intracellular ATP content, was altered compared to the parental strain.

Bacterial cell surface charge is responsible for susceptibility to cationic antimicrobial peptides. Previously, Staphylococcus aureus dlt and mprF were identified as factors conferring a positive charge upon cell surfaces. In this study, we investigated the regulation of cell surface charge during growth. Using a group of S. aureus MW2 mutants, which are gene-inactivated in 15 types of two-component systems (TCSs), we tested dltC and mprF expression and found that two TCSs, aps and agr, were associated with dltC and mprF expression in a growth phase-dependent manner. The first of these, aps, which had already been identified as a sensor of antimicrobial peptides and a positive regulator of dlt and mprF expression, was expressed strongly in the exponential phase, while its expression was significantly suppressed by agr in the stationary phase, resulting in higher expression of dltC and mprF in the exponential phase and lower expression in the stationary phase. Since both types of expression affected the cell surface charge, the susceptibility to antimicrobial peptides and cationic antibiotics was changed during growth. Furthermore, we found that the ability to sense antimicrobial peptides only functioned in the exponential phase. These results suggest that cell surface charge is tightly regulated during growth in S. aureus.

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C. albicans interactions with endothelial cells. Using an in vitro flow adhesion assay, we previously observed that yeast cells bind in greater numbers to human microvascular endothelial cells than do hyphal or pseudohyphal cells. This is consistent with previous observations that, in a murine model of disseminated candidiasis, cells locked in the yeast form can efficiently escape the bloodstream and invade host tissues. To more precisely explore the role of Als3p in adhesion and virulence, we deleted both copies of ALS3 in a wild-type C. albicans strain. In agreement with previous studies, our als3Δ null strain formed hyphae normally but was defective in biofilm formation. Whilst ALS3 was not expressed in our null strain, hypha-specific genes such as ECE1 and HWP1 were still induced appropriately. Both the yeast form and the hyphal form of the als3Δ strain adhered to microvascular endothelial cells to the same extent as a wild-type strain under conditions of flow, indicating that Als3p is not a significant mediator of the initial interaction between fungal cells and the endothelium. Finally, in a murine model of haematogenously disseminated candidiasis the mutant als3Δ remained as virulent as the wild-type parent strain.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo–in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to PII protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na+/H+ antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.