Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages, Providing New Insights into the Evolution of EHEC.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the "big five"), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

Two distinct coagulase-dependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model.

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options.

Unique virulence properties of Yersinia enterocolitica O:3--an emerging zoonotic pathogen using pigs as preferred reservoir host.

Enteropathogenic Yersinia enterocolitica bioserotype 4/O:3 are the most frequent cause of human yersiniosis worldwide with symptoms ranging from mild diarrhea to severe complications of mesenteric lymphadenitis, liver abscesses and postinfectious extraintestinal sequelae. The main reservoir host of 4/O:3 strains are pigs, which represent a substantial disease-causing potential for humans, as they are usually asymptomatic carriers. Y. enterocolitica O:3 initiates infections by tight attachment to the intestinal mucosa. Colonization of the digestive tract is frequently followed by invasion of the intestinal layer primarily at the follicle-associated epithelium, allowing the bacteria to propagate in the lamina propria and disseminate into deeper tissues. Molecular characterization of Y. enterocolitica O:3 isolates led to the identification of (i) alternative virulence and fitness factors and (ii) small genetic variations which cause profound changes in their virulence gene expression pattern (e.g. constitutive expression of the primary invasion factor InvA). These changes provoke a major difference in the virulence properties, i.e. reduced colonization of intestinal tissues in mice, but improved long-term colonization in the pig intestine. Y. enterocolitica O:3 strains cause also a considerably lower level of proinflammatory cytokine IL-8 and higher levels of the anti-inflammatory cytokine IL-10 in porcine primary macrophages, as compared to murine macrophages, which could contribute to limiting inflammation, immunopathology and severity of the infection in pigs.

A molecular scheme for Yersinia enterocolitica patho-serotyping derived from genome-wide analysis.

Yersinia enterocolitica is a food-borne, gastro-intestinal pathogen with world-wide distribution. Only 11 serotypes have been isolated from patients, with O:3, O:9, O:8 and O:5,27 being the serotypes most commonly associated with human yersiniosis. Serotype is an important characteristic of Y. enterocolitica strains, allowing differentiation for epidemiology, diagnosis and phylogeny studies. Conventional serotyping, performed by slide agglutination, is a tedious and laborious procedure whose interpretation tends to be subjective, leading to poor reproducibility. Here we present a PCR-based typing scheme for molecular identification and patho-serotyping of Y. enterocolitica. Genome-wide comparison of Y. enterocolitica sequences allowed analysis of the O-antigen gene clusters of different serotypes, uncovering their formerly unknown genomic locations, and selection of targets for serotype-specific amplification. Two multiplex PCRs and one additional PCR were designed and tested on various reference strains and isolates from different origins. Our genotypic assay proved to be highly specific for identification of Y. enterocolitica species, discrimination between virulent and non-virulent strains, distinguishing the main human-related serotypes, and typing of conventionally untypeable strains. This genotyping scheme could be applied in microbiology laboratories as an alternative or complementary method to the traditional phenotypic assays, providing data for epidemiological studies.

Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment.

Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118-130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment.

Rabbit monoclonal antibodies directed at the T3SS effector protein YopM identify human pathogenic Yersinia isolates.

The Yersinia outer protein M (YopM) is a type 3 secretion system (T3SS)-dependent effector protein of Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Although YopM is indispensable for full virulence, its molecular functions still remain largely elusive. Recently, we could identify the recombinant YopM (rYopM) protein derived from the Y. enterocolitica strain 8081 (JB580) as a cell-penetrating protein, which down-regulates the expression of various pro-inflammatory cytokines including TNFα. In this study, we have generated rabbit monoclonal anti-YopM antibodies (RabMabs). RabMabs were characterized by SDS-PAGE and Western blotting using various truncated versions of rYopM to identify epitope-containing domains. RabMabs recognizing either the N- or C-terminus of YopM were characterized further and validated using a collection of 61 pathogenic and non-pathogenic Yersinia strains as well as exemplary strains of major intestinal bacterial pathogens such as Salmonella enterica ssp. enterica, Shigella flexneri and intestinal pathogenic Escherichia coli. RabMab 41.3 directed at the N-terminus of YopM of Y. enterocolitica strain 8081 recognized all YopM-expressing pathogenic Yersinia strains analyzed in this study but failed to recognize non-pathogenic isolates. Thus, RabMab 41.3 might be applicable for the detection of pathogenic Yersinia strains.

Microevolution of Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung.

Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistance of P. aeruginosa in the CF lung requires a sophisticated habitat-specific adaptation of this pathogen to the heterogeneous and fluctuating lung environment. Due to the high selective pressure of inflamed CF lungs, P. aeruginosa increasingly experiences complex physiological and morphological changes. Pulmonary adaptation of P. aeruginosa is mediated by genetic variations that are fixed by the repeating interplay of mutation and selection. In this context, the emergence of hypermutable phenotypes (mutator strains) obviously improves the microevolution of P. aeruginosa to the diverse microenvironments of the CF lung. Mutator phenotypes are amplified during CF lung disease and accelerate the intraclonal diversification of P. aeruginosa. The resulting generation of numerous subclonal variants is advantegous to prepare P. aeruginosa population for unpredictable stresses (insurance hypothesis) and thus supports long-term survival of this pathogen. Oxygen restriction within CF lung environment further promotes persistence of P. aeruginosa due to increased antibiotic tolerance, alginate production and biofilm formation. Finally, P. aeruginosa shifts from an acute virulent pathogen of early infection to a host-adapted chronic virulent pathogen of end-stage infection of the CF lung. Common changes that are observed among chronic P. aeruginosa CF isolates include alterations in surface antigens, loss of virulence-associated traits, increasing antibiotic resistances, the overproduction of the exopolysaccharide alginate and the modulation of intermediary and micro-aerobic metabolic pathways (Hogardt and Heesemann, Int J Med Microbiol 300(8):557-562, 2010). Loss-of-function mutations in mucA and lasR genes determine the transition to mucoidity and loss of quorum sensing, which are hallmarks of the chronic virulence potential of P. aeruginosa. Metabolic factors that are positively selected in response to the specific environment of CF lung include the outer membrane protein OprF, the microaerophilic oxidase Cbb3-2, the blue copper protein azurin, the cytochrome c peroxidase c551 and the enzymes of the arginine deiminase pathway ArcA-ArcD. These metabolic adaptations probably support the growth of P. aeruginosa within oxygen-depleted CF mucus. The deeper understanding of the physiological mechanisms of niche specialization of P. aeruginosa during CF lung infection will help to identify new targets for future anti-pseudomonal treatment strategies to prevent the selection of mutator isolates and the establishment of chronic CF lung infection.

A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 µM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.

Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis.

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.

Yersinia enterocolitica inactivates NK cells.

Natural Killer (NK) cells serve as an important source of proinflammatory cytokines early during infection. Hypothesizing that Yersinia enterocolitica might interact with and inactivate NK cells, we examined NK cell-Y. enterocolitica interactions in vitro and in vivo. Y. enterocolitica adheres to NK cells in an Invasin dependent manner and inhibits NK cell cytotoxicity and IFN-γ production induced by IL-12+IL-18 or IL-12 alone. YopP, an acetyltransferase known to inhibit MAPK and NFκB signaling, suppresses IL-12 and IL-12+IL-18 mediated IFN-γ production in NK cells by inhibiting phosphorylation of Tyk2 and STAT4 in addition to MAPK. YopP inhibits induction of all genes whose expression is induced by IL-12+IL-18 in NK cells. Y. enterocolitica-mediated adherence to and inactivation of NK cells also occurs after infection in vivo. Thus, we present the first report of a bacterial pathogen inactivating NK cells, and report interaction with Tyk2-STAT4 signaling as a novel function of YopP.

Yersinia enterocolitica and Photorhabdus asymbiotica ß-lactamases BlaA are exported by the twin-arginine translocation pathway.

In general, ß-lactamases of medically important Gram-negative bacteria are Sec-dependently translocated into the periplasm. In contrast, ß-lactamases of Mycobacteria spp. (BlaC, BlaS) and the Gram-negative environmental bacteria Stenotrophomonas maltophilia (L2) and Xanthomonas campestris (Bla(XCC-1)) have been reported to be secreted by the twin-arginine translocation (Tat) system. Yersinia enterocolitica carries 2 distinct ß-lactamase genes (blaA and blaB) encoding BlaA(Ye) and the AmpC-like ß-lactamase BlaB, respectively. By using the software PRED-TAT for prediction and discrimination of Sec from Tat signal peptides, we identified a functional Tat signal sequence for Yersinia BlaA(Ye). The Tat-dependent translocation of BlaA(Ye) could be clearly demonstrated by using a Y. enterocolitica tatC-mutant and cell fractionation. Moreover, we could demonstrate a unique unusual temperature-dependent activity profile of BlaA(Ye) ranging from 15 to 60 °C and a high 'melting temperature' (T(M)=44.3°) in comparison to the related Sec-dependent ß-lactamase TEM-1 (20-50°C, T(M)=34.9 °C). Strikingly, the blaA gene of Y. enterocolitica is present in diverse environmental Yersinia spp. and a blaA homolog gene could be identified in the closely related Photorhabdus asymbiotica (BlaA(Pa); 69% identity to BlaA(Ye)). For BlaA(Pa) of P. asymbiotica, we could also demonstrate Tat-dependent secretion. These results suggest that Yersinia BlaA-related ß-lactamases may be the prototype of a large Tat-dependent ß-lactamase family, which originated from environmental bacteria.

Novel highly sensitive IL-10-beta-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo.

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme ß-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-ß-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.

Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin.

Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a ß1 integrin-dependent adherence with subsequent internalization of yersiniae by a ß1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine ß1 integrin-expressing cell lines we could demonstrate the role of the ß1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human ß1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that ß1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human ß1 integrins.

The Yersinia enterocolitica type 3 secretion system (T3SS) as toolbox for studying the cell biological effects of bacterial Rho GTPase modulating T3SS effector proteins.

The bacterial effector proteins IpgB(1) and IpgB(2) of Shigella and Map of Escherichia coli activate the Rho GTPases Rac1, RhoA and Cdc42, respectively, whereas YopE and YopT of Yersinia inhibit these Rho family GTPases. We established a Yersinia toolbox which allows to study the cellular effects of these effectors in different combinations in the context of Yersinia type 3 secretion system (Ysc)-T3SS-mediated injection into HeLa cells. For this purpose hybrid proteins were constructed by fusion of YopE with the effector protein of interest. As expected, injected hybrid proteins induced membrane ruffles and Yersinia uptake for IpgB(1) , stress fibres for IpgB(2) and microspikes for Map. By co-infection experiments we could demonstrate (i) IpgB(2) -mediated and ROCK-dependent inhibition of IpgB(1) -mediated Rac1 effects, (ii) YopT-mediated suppression of IpgB(1) -induced Yersinia invasion and (iii) failure of YopE-mediated suppression of IpgB(1) -induced Yersinia invasion, presumably due to preferential inhibition of RhoG by YopE GAP function. By infecting polarized MDCK cells we could demonstrate that Map or IpgB(1) but not IpgB(2) affects cell monolayer integrity. In summary, the Yersinia toolbox is suitable to study cellular effects of effector proteins of diverse bacterial species separately or in combination in the context of bacterial T3SS-mediated injection.

Link Between Antibiotic Persistence and Antibiotic Resistance in Bacterial Pathogens.

Both, antibiotic persistence and antibiotic resistance characterize phenotypes of survival in which a bacterial cell becomes insensitive to one (or even) more antibiotic(s). However, the molecular basis for these two antibiotic-tolerant phenotypes is fundamentally different. Whereas antibiotic resistance is genetically determined and hence represents a rather stable phenotype, antibiotic persistence marks a transient physiological state triggered by various stress-inducing conditions that switches back to the original antibiotic sensitive state once the environmental situation improves. The molecular basics of antibiotic resistance are in principle well understood. This is not the case for antibiotic persistence. Under all culture conditions, there is a stochastically formed, subpopulation of persister cells in bacterial populations, the size of which depends on the culture conditions. The proportion of persisters in a bacterial population increases under different stress conditions, including treatment with bactericidal antibiotics (BCAs). Various models have been proposed to explain the formation of persistence in bacteria. We recently hypothesized that all physiological culture conditions leading to persistence converge in the inability of the bacteria to re-initiate a new round of DNA replication caused by an insufficient level of the initiator complex ATP-DnaA and hence by the lack of formation of a functional orisome. Here, we extend this hypothesis by proposing that in this persistence state the bacteria become more susceptible to mutation-based antibiotic resistance provided they are equipped with error-prone DNA repair functions. This is - in our opinion - in particular the case when such bacterial populations are exposed to BCAs.

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3.

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).

Yersinia enterocolitica palearctica serobiotype O:3/4--a successful group of emerging zoonotic pathogens.

BACKGROUND: High-pathogenic Y. enterocolitica ssp. enterocolitica caused several human outbreaks in Northern America. In contrast, low pathogenic Y. enterocolitica ssp. palearctica serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three Y. enterocolitica ssp. palearctica serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic Y. enterocolitica ssp. enterocolitica 8081 O:8/1B to address the peculiarities of the O:3/4 group. RESULTS: Most high-pathogenicity-associated determinants of Y. enterocolitica ssp. enterocolitica (like the High-Pathogenicity Island, yts1 type 2 and ysa type 3 secretion systems) are absent in Y. enterocolitica ssp. palearctica serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different ysp type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (aga-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the Y. enterocolitica ssp. palearctica serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the aga-operon to support the growth of the Y. enterocolitica ssp. enterocolitica O:8/1B on GalNAc. CONCLUSIONS: Y. enterocolitica ssp. palearctica serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.

Farnesol misplaces tip-localized Rho proteins and inhibits cell wall integrity signalling in Aspergillus fumigatus.

Farnesol is known for inducing apoptosis in some fungi and mammalian cells. To evaluate its potential role as an antifungal agent, we studied its impact on the human pathogen Aspergillus fumigatus. We found that growth of A. fumigatus wild type is inhibited, but two cell wall mutants, Deltamnt1 andDeltaglfA, are much more susceptible to farnesol. This susceptibility is partially rescued by osmotic stabilization, suggesting that farnesol is a cell wall perturbing agent. However, farnesol does not activate but inhibit the cell wall integrity (CWI) pathway. Remarkably, mutants lacking AfMkk2 or AfMpkA, two kinases essential for CWI signalling, are also highly susceptible to farnesol, suggesting that its mode of action goes beyond inhibition of CWI signalling. Farnesyl derivatives are known for interfering with the function of prenylated proteins. We analysed the subcellular localization of two prenylated Rho family GTPases, AfRho1 and AfRho3, which are implicated in controlling CWI and the cytoskeleton. We found that under normal growth conditions AfRho1 and AfRho3 predominantly localize to the hyphal tip. After farnesol treatment this localization is rapidly lost, which is accompanied by swelling of the hyphal tips. Parallel displacement of tropomyosin from the tips suggests a concomitant disorganization of the apical actin cytoskeleton.

Interleukin-1 receptor-associated kinase-M suppresses systemic lupus erythematosus.

OBJECTIVES: Interleukin-1 receptor-associated kinase (IRAK)-M suppresses Toll-like receptor (TLR)-mediated activation of innate immunity during infection. A similar role was hypothesised for IRAK-M in autoimmunity. METHODS: Irak-m-deficient mice were crossed with autoimmune C57BL/6-lpr/lpr mice and detailed phenotype analysis was performed. RESULTS: Irak-m deficiency converted the mild autoimmune phenotype of C57BL/6-lpr/lpr mice into a massive lymphoproliferative syndrome with lethal autoimmune lung disease and lupus nephritis. Irak-m deficiency induced a number of interferon-related genes, cytokines and plasma cell survival factors in spleen cells of these mice. Irak-m-deficient C57BL/6-lpr/lpr mice showed expansion of autoreactive T cells, dysfunctional regulatory T cells and plasma cells which was associated with increased lupus autoantibody production. TLR7 antagonism almost completely abrogated this phenotype consistent with IRAK-M-mediated suppression of TLR7 signalling in vitro. CONCLUSIONS: These data identify a previously unknown function of IRAK-M-namely, suppression of TLR7-mediated autoimmunity-and mutant IRAK-M as a previously unknown genetic risk for murine SLE.