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Abstract Background The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation. Results In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation. Conclusions Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated throughout the staphylococci.

Abstract Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

Abstract Background Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant. Results We observed that flagellin was consistently and strongly present in the secretome of wild type EPEC and the ΔespADB mutant but present only weakly in the secretome of the ΔescF mutant. Given the ancestral relationship between the flagella export apparatus and virulence associated T3SSs, we investigated whether FliC could utilise the LEE-encoded T3SS for export. In the absence of a functional flagella export apparatus, we showed that FliC could be secreted by the LEE-encoded T3SS and stimulate (Toll-like receptor 5) TLR5 signalling but could not confer motility. Conclusion Since the secretion of FliC during A/E lesion formation would presumably be disadvantageous for the pathogen, we propose that virulence associated T3SSs and flagella T3SSs have evolved through a system of chaperones and complex regulatory pathways to be functional at different times to ensure that FliC secretion does not occur during T3SS effector translocation.

Abstract Background Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure. Results We have performed a fresh bioinformatics analysis of the LEE. Using PSI-BLAST we have been able to identify several novel homologies between LEE-encoded and Ysc-Yop-associated proteins: Orf2/YscE, Orf5/YscL, rORF8/EscI, SepQ/YscQ, SepL/YopN-TyeA, CesD2/LcrR. In addition, we highlight homology between EspA and flagellin, and report many new homologues of the chaperone CesT. Conclusion We conclude that the vast majority of LEE-encoded proteins do indeed possess homologues and that homology data can be used in combination with experimental data to make fresh functional predictions.