- Volume 154, Issue 5, 2008

Nectin-1, a member of the immunoglobulin superfamily, is a Ca2+-independent cell adhesion protein implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also regulates cell–cell adhesion and cell polarization in a Cdc42- and Rac-dependent manner. Western blot analyses demonstrated that accumulation of host nectin-1 is decreased by 85 % at 48 hours post-infection (h.p.i.) in Chlamydia trachomatis serovar E-infected HeLa cells. Time-course experiments demonstrated that this decrease was sustained to 60 h.p.i. Nectin-1 downregulation in C. trachomatis-infected cells was prevented by both chloramphenicol exposure and prior inactivation of the chlamydiae with UV light, demonstrating that active C. trachomatis replication was required. Penicillin G-exposure studies demonstrated that nectin-1 accumulation was also altered during persistent infection. Finally, RT-PCR analyses indicated that chlamydial infection did not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 accumulation is reduced at a post-transcriptional level. Intesrestingly, N-cadherin-dependent cell–cell junctions can be disrupted by C. trachomatis infection, as reported by Prozialeck et al. (2002) . Because interaction of nectin molecules on adjacent cells is essential for AJ formation, these data suggest that C. trachomatis may disrupt AJs, at least in part, by diminishing nectin-1 accumulation. Notably, release of chlamydiae-infected epithelial cells has been observed both in vitro from polarized monolayers and in vivo from tissues, suggesting that chlamydia-modulated downregulation of adhesion molecules and the subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis.

Ureaplasma species (Ureaplasma parvum and Ureaplasma urealyticum) are commonly isolated pathogens from the female reproductive tract and are associated with perinatal diseases in humans. Inappropriate induction of inflammatory responses may be involved in the occurrence of such diseases; however, pathogenic agents that induce the inflammatory response have not been identified in ureaplasmas. In this study, we examined the involvement of Toll-like receptors (TLRs) in the activation of the immune response by U. parvum lipoproteins, as well as the U. parvum components responsible for nuclear factor κB (NF-κB) activation. The Triton X-114 (TX-114) detergent phase of U. parvum was found to induce NF-κB through TLR2. The active components of the TX-114 detergent phase were lipoproteins, such as multiple banded (MB) antigen, UU012 and UU016 of U. parvum. The activation of NF-κB by these lipoproteins was inhibited by dominant negative (DN) constructs of TLR1 and DN TLR6. Thus, the lipoproteins from U. parvum were found to activate NF-κB through TLR1, TLR2 and TLR6. Furthermore, these lipoproteins possessed an ability to induce tumour necrosis factor-alpha (TNF-α) in mouse peritoneal macrophages.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

We have previously shown that copper uptake and regulation in the opportunistic pathogen Candida albicans has some similarities to those in Saccharomyces cerevisiae, including the activation of the copper transporter gene CaCTR1 under low-copper conditions by the transcription factor CaMac1p. However, in this study, further analysis has shown that the actual mechanism of regulation by CaMac1p is different from that of its S. cerevisiae homologue. We demonstrate for the first time, to our knowledge, that the CaMAC1 gene is transcriptionally autoregulated in a copper-dependent manner, in contrast to ScMAC1, which is constitutively transcribed. We also demonstrate that the presence of one copper response element in the promoters of CaCTR1, CaMAC1 and the ferric/cupric reductase gene CaFRE7 is sufficient for normal levels of copper-responsive transcription. In contrast, two promoter elements are essential for normal levels of copper-dependent transcriptional activation by ScMac1p. CaMac1p is also involved in the regulation of the iron-responsive transcriptional repressor gene SFU1 and the alternative oxidase gene AOX2. This work describes a key feature of the copper uptake system in C. albicans that distinguishes it from similar processes in the model yeast S. cerevisiae. The importance of copper uptake in the environment of the human host and the implications for the disease process are discussed.

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 °C were 0.045 h−1, 0.06 h−1 and 0.055 h−1, respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h−1, 0.1 h−1 and 0.17 h−1, respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)−1, 0.53 g dm (g ETBE)−1, 0.81 g dm (g TAME)−1, 0.48 g dm (g TBA)−1, 0.76 g dm (g TAA)−1 and 0.54 g dm (g 2-HIBA)−1 were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h−1 (g dm)−1, 1.11 mmol ETBE h−1 g−1, 0.66 mmol TAME h−1 g−1, 1.19 mmol TAA h−1 g−1, 2.82 mmol TBA h−1 g−1, and 3.27 mmol 2-HIBA h−1 g−1. The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 °C, but at 5 °C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 °C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.

In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling.