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.

Type II secretion in Yersinia-a secretion system for pathogenicity and environmental fitness.

In Yersinia species, type III secretion (T3S) is the most prominent and best studied secretion system and a hallmark for the infection process of pathogenic Yersinia species. Type II secretion (T2S), on the other hand, is less well-characterized, although all Yersinia species, pathogenic as well as non-pathogenic, possess one or even two T2S systems. The only Yersinia strain in which T2S has so far been studied is the human pathogenic strain Y. enterocolitica 1b. Mouse infection experiments showed that at least one of the two T2S systems of Y. enterocolitica 1b, termed Yts1, is involved in dissemination and colonization of deeper tissues like liver and spleen. Interestingly, in vitro studies revealed a complex regulation of the Yts1 system, which is mainly active at low temperatures and high Mg(2+)-levels. Furthermore, the functional characterization of the proteins secreted in vitro indicates a role of the Yts1 machinery in survival of the bacteria in an environmental habitat. In silico analyses identified Yts1 homologous systems in bacteria that are known as plant symbionts or plant pathogens. Thus, the recent studies point to a dual function of the Yts1 T2S systems, playing a role in virulence of humans and animals, as well as in the survival of the bacteria outside of the mammalian host. In contrast, the role of the second T2S system, Yts2, remains ill defined. Whereas the T3S system and its virulence-mediating role has been intensively studied, it might now be time to also focus on the T2S system and its role in the Yersinia lifestyle, especially considering that most of the Yersinia isolates are not found in infected humans but have been gathered from various environmental samples.

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells.

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1­7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6­7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6­7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.