- Volume 154, Issue 11, 2008

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1–10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Δsap1–3, Δsap4–6, Δefg1 and Δefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4–6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Δsap1–3, expression was compensated for by increased expression of SAP5, and in Δsap4–6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Δsap1–3 and Δsap4–6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Δefg1/cph1 and Δefg1, but SAP5 expression was only dramatically reduced in Δefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1–3 and Sap4–6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.

A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1–SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1–SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1–SAP3 or the SAP4–SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Δ mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p–Sap6p are not required for invasion of RHE by C. albicans.

We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P2 levels and the cell integrity pathway, and that PI(4,5)P2 homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.

Candida glabrata undergoes reversible, high-frequency core switching between phenotypes that include dark brown (DB), light brown (LB) and white (Wh). These phenotypes in turn can switch to the irregular wrinkle (IWr) phenotype. Natural isolates, however, express predominantly the DB phenotype, leading to the hypothesis that it has a colonization advantage over the other switch phenotypes. Using the mouse model of systemic infection, results are presented which support this hypothesis. DB has an advantage over other switch phenotypes in colonizing the two major target organs in the mouse model, the spleen and liver. A time-course study reveals that colonization of the major target organs occurs very rapidly (within 2 h) after host injection, and that the DB advantage for spleen and liver colonization is immediate. The DB advantage is maintained during clearing from spleen, liver and kidneys, and during delayed transient brain colonization. These results demonstrate that DB has a colonization advantage over other switch phenotypes, and that the switch phenotype expressed by a colonizing population therefore plays a fundamental role in virulence. It is therefore essential that switching be considered in both in vivo and in vitro studies of C. glabrata virulence.

The surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID50, but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID50 increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.

Three Mycobacterium tuberculosis proteins, PE_PGRS 16 (Rv0977), PE_PGRS 26 (Rv1441c) and PE_PGRS 33 (Rv1818c), were expressed in Mycobacterium smegmatis and used to investigate the host response to members of this unique protein family. Following infection of macrophages with the recombinant M. smegmatis (Ms) strains, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 were significantly more persistent (4.4 and 4.2 log c.f.u.) compared with Ms-PE_PGRS 16 (3.4 log c.f.u.) at day 6. Similarly, after infection of mice, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 persisted at significantly higher levels in the spleen (3.5 and 3.2 log c.f.u.) and liver (3 and 2.6 log c.f.u.) compared with Ms-PE_PGRS 16 in the spleen (2 log c.f.u.) and in the liver (1 log c.f.u.) at day 10. Increased persistence of Ms-PE_PGRS 33 and Ms-PE_PGRS 26 was associated with cell death and increased release of lactate dehydrogenase in macrophage cultures as well as increased levels of IL-10 and, in contrast, lower levels of IL-12 and NO both in vitro and in mouse splenocytes. Conversely, poor survival of Ms-PE_PGRS 16 was associated both in macrophage cultures and in vivo with higher levels of NO and IL-12. All three PE_PGRS proteins were found to be cell-surface antigens, but immunization of mice with these PE_PGRS antigens as DNA vaccines showed no protection in a TB aerosol challenge model. In general, the results suggest that variable expression of different PE_PGRS proteins within host cells can affect either the fate of the mycobacterial pathogen or that of the host during infection and point to the importance of studying the expression and function of individual members of the PE_PGRS gene family of M. tuberculosis.

Staphylococcus aureus is an important nosocomial and community-acquired pathogen. Hospital infections are frequently complicated by the ability of bacteria to form biofilms on different surfaces. The development of bacterial films on medical indwelling devices, such as prostheses, often requires surgical procedures to remove the contaminated implant. Indeed, biofilm formation on central endovenous catheters is a major cause of primary bacteraemia in hospitals. The modulation of virulence factors in S. aureus is orchestrated by a number of global regulators including agr RNAIII. To improve our understanding of the role of the agr quorum-sensing system in biofilm formation by S. aureus, we constructed a number of agr-null mutants, derived from contemporary clinical isolates. Analysis of these mutants indicates that agr has a significant impact on biofilm development for most of the isolates tested. Our data show that RNAIII can control both biofilm formation and accumulation. The agr effect included both up- and downregulation of biofilms, even for isolates within the same lineage, corroborating the hypothesis that the mechanisms involved in S. aureus biofilms are complex and probably multifactorial.

SpiC is a virulence factor encoded within Salmonella pathogenicity island 2 (SPI-2). We have previously reported that infection of macrophages with Salmonella enterica serovar Typhimurium results in the SPI-2-dependent activation of the mitogen-activated protein kinase (MAPK) signalling pathways, leading to the expression of suppressor of cytokine signalling (SOCS)-3, which is involved in the inhibition of cytokine signalling. Here, we investigated the mechanism by which SpiC mediates the activation of signal transduction pathways in macrophages. Proteomic analysis showed that the level of FliC protein, a component of the flagellar filaments, was lower in the culture supernatant of a spiC mutant than in the supernatant from wild-type Salmonella. Furthermore, quantitative real-time RT-PCR showed that this mutant had a much lower level of fliC mRNA, indicating that SpiC regulates the transcription of FliC. We also found that the level of SOCS-3 in J774 macrophages was lower when they were infected with the fliC mutant than with wild-type Salmonella. FliC participated in the activation of MAPK pathways in Salmonella-infected macrophages, leading to the upregulation of SOCS-3 expression. Collectively, these results indicate that SpiC is involved in the expression of FliC, which plays a significant role in the SPI-2-dependent activation of MAPK pathways in Salmonella-infected macrophages.

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode ∼1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 °C and 37 °C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

Cyt1Aa is a δ-endotoxin protein that is produced by Bacillus thuringiensis subsp. israelensis. It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23–24 kDa and a threefold increase in activity. In this research, Cyt1Aa crystals were purified from B. thuringiensis IPS78/11 harbouring the expression vector pHT-cyAp20. The activity of the activated form of Cyt1Aa (23–24 kDa) was examined on a pathogenic strain of the Gram-negative Escherichia coli and the Gram-positive species Staphylococcus aureus. The Cyt1Aa minimal inhibitory concentration for E. coli and S. aureus was 1.25 and 5 μg ml−1, respectively. Cyt1Aa was found to be bactericidal for E. coli, whereas it was bacteriostatic for S. aureus. Furthermore, Cyt1Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole δ-endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1Aa has a bactericidal effect upon Gram-negative bacteria.

Survival strategies exhibited over 4 years by Ralstonia solanacearum phylotype (ph) II biovar (bv) 2 in environmental water microcosms were examined. The bacterium is a devastating phytopathogen whose ph II bv 2 causes bacterial wilt in solanaceous crops and ornamental plants. Outbreaks of the disease may originate from dissemination of the pathogen in watercourses, where it has to cope with prolonged nutrient limitation. To ascertain the effect of long-term starvation on survival and pathogenicity of R. solanacearum in natural water microcosms, survival experiments were conducted. Microcosms were prepared from different sterile river water samples, inoculated separately with two European strains of ph II at 106 c.f.u. ml−1 and maintained at 24 °C for 4 years. In all assayed waters, starved R. solanacearum remained in a non-growing but culturable state during the first year, maintaining approximately the initial numbers. Thereafter, part of the population of R. solanacearum progressively lost the ability to form colonies, and non-culturable but metabolically active cells appeared. During the whole period, the bacterium remained pathogenic on host plants and underwent a transition from typical bacilli to small cocci which tended to aggregate. Some starved R. solanacearum cells filamented and formed buds. Starvation response, viable but non-culturable state, morphological changes and aggregation have not previously been reported for this pathogen as survival mechanisms induced in oligotrophic conditions. The potential existence of long-starved pathogenic cells in environmental waters may raise new concerns about the epidemiology of bacterial wilt disease.

Rhodotorula glutinis IFO1125 was found to acquire increased aluminium (Al) resistance from 50 μM to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. To investigate the mechanism underlying this novel phenomenon, wild-type and Al-resistant cells were compared. Neither cell type accumulated the free form of Al (Al3+) added to the medium. Transmission electron microscopic analyses revealed a greater number of mitochondria in resistant cells. The formation of small mitochondria with simplified cristae structures was observed in the wild-type strain grown in the presence of Al and in resistant cells grown in the absence of Al. Addition of Al to cells resulted in high mitochondrial membrane potential and concomitant generation of reactive oxygen species (ROS). Exposure to Al also resulted in elevated levels of oxidized proteins and oxidized lipids. Addition of the antioxidants α-tocopherol and ascorbic acid alleviated the Al toxicity, suggesting that ROS generation is the main cause of Al toxicity. Differential display analysis indicated upregulation of mitochondrial genes in the resistant cells. Resistant cells were found to have 2.5- to 3-fold more mitochondrial DNA (mtDNA) than the wild-type strain. Analysis of tricarboxylic acid cycle and respiratory-chain enzyme activities in wild-type and resistant cells revealed significantly reduced cytochrome c oxidase activity and resultant high ROS production in the latter cells. Taken together, these data suggest that the adaptive increased resistance to Al stress in resistant cells resulted from an increased number of mitochondria and increased mtDNA content, as a compensatory response to reduced respiratory activity caused by a deficiency in complex IV function.

We have studied the role of the pga1 gene of Penicillium chrysogenum, encoding the alpha subunit of a heterotrimeric G protein, in secondary metabolite production. The dominant activating pga1G42R mutation caused an increase in the production of the three secondary metabolites penicillin, the yellow pigment chrysogenin and the mycotoxin roquefortine, whereas the dominant inactivating pga1G203R allele and the deletion of the pga1 gene resulted in a decrease of the amount of produced penicillin and roquefortine. Chrysogenin is produced in solid medium as a yellow pigment, and its biosynthesis is clearly enhanced by the presence of the dominant activating pga1G42R allele. Roquefortine is produced associated with mycelium during the first 3 days in submerged cultures, and is released to the medium afterwards; dominant activating and inactivating pga1 mutations result in upregulation and downregulation of roquefortine biosynthesis recpectively. Pga1 regulates penicillin biosynthesis by controlling expression of the penicillin biosynthetic genes; the three genes pcbAB, pcbC and penDE showed elevated transcript levels in transformants expressing the pga1G42R allele, whereas in transformants with the inactivating pga1G203R allele and in the pga1-deleted mutant their transcript levels were lower than those in the parental strains. Increase of intracellular cAMP levels had no effect on penicillin production. In summary, the dominant activating pga1G42R allele upregulates the biosynthesis of three secondary metabolites in Penicillium chrysogenum to a different extent.