- Volume 155, Issue 9, 2009

Enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 is naturally exposed to a wide variety of stresses including gastric acid shock, and yet little is known about how this stress influences virulence. This study investigated the impact of acid stress on several critical virulence properties including survival, host adhesion, Shiga toxin production, motility and induction of host-cell apoptosis. Several acid-stress protocols with relevance for gastric passage as well as external environmental exposure were included. Acute acid stress at pH 3 preceded by acid adaptation at pH 5 significantly enhanced the adhesion of surviving organisms to epithelial cells and bacterial induction of host-cell apoptosis. Motility was also significantly increased after acute acid stress. Interestingly, neither secreted nor periplasmic levels of Shiga toxin were affected by acid shock. Pretreatment of bacteria with erythromycin eliminated the acid-induced adhesion enhancement, suggesting that de novo protein synthesis was required for the enhanced adhesion of acid-shocked organisms. DNA microarray was used to analyse the transcriptome of an EHEC O157 : H7 strain exposed to three different acid-stress treatments. Expression profiles of acid-stressed EHEC revealed significant changes in virulence factors associated with adhesion, motility and type III secretion. These results document profound changes in the virulence properties of EHEC O157 : H7 after acid stress, provide a comprehensive genetic analysis to substantiate these changes and suggest strategies that this pathogen may use during gastric passage and colonization in the human gastrointestinal tract.

Toll-like receptor 4 (TLR4) senses bacterial LPS and is required for the control of systemic Salmonella enterica serovar Typhimurium infection in mice. The mechanisms of TLR4 activation and its downstream signalling cascades are well described, yet the direct effects on the pathogen of signalling via this receptor remain unknown. To investigate this we used microarray-based transcriptome profiling of intracellular S. Typhimurium during infection of primary bone marrow-derived macrophages from wild-type and TLR4-deficient mice. We identified 17 S. Typhimurium genes that were upregulated in the presence of functional TLR4. Nine of these genes have putative functions in oxidative stress resistance. We therefore examined S. Typhimurium gene expression during infection of NADPH oxidase-deficient macrophages, which lack normal oxidative killing mechanisms. We identified significant overlap between the ‘TLR4-responsive’ and ‘NADPH oxidase-responsive’ genes. This is new evidence for a link between TLR4 signalling and NADPH oxidase activity. Interestingly, with the exception of a dps mutant, S. Typhimurium strains lacking individual TLR4- and/or oxidative stress-responsive genes were not attenuated during intravenous murine infections. Our study shows that TLR4 activity, either directly or indirectly, induces the expression of multiple stress resistance genes during the intracellular life of S. Typhimurium.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C6 or C2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the ΔilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the ΔilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, one of the most serious rice diseases. The xrvA gene from Xoo strain 13751 encodes a protein containing a histone-like nucleoid-structuring protein (H-NS) domain. The expression of xrvA in strain 13751 was enhanced in XOM2 minimal medium. Mutation of the xrvA gene of strain 13751 led to a significant reduction in virulence in the host plant rice, a delayed hypersensitive response in the nonhost castor-oil plant, a decrease in extracellular polysaccharide and diffusible signal factor production, and an increase in intracellular glycogen accumulation. Northern hybridization analyses revealed that the virulence-associated genes hrpG, hrpX, rpfC, rpfF, rpfG and gumB were downregulated in the xrvA mutant compared to the wild-type and complemented strains. Interestingly, increase of copy number of xrvA in the wild-type strain 13751 resulted in a strain showing similar phenotypes as the xrvA mutant and a reduction of the expression of gumB, hrpX, rpfC, rpfF and rpfG. These findings indicate that the xrvA gene, which is highly conserved in the sequenced strains of Xanthomonas, encodes an important regulatory factor for the virulence of Xoo.

Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, α-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and α-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and α-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is the causative agent of common sexually transmitted diseases and the leading cause of preventable blindness worldwide. It has been observed that YtgA (CT067) is very immunogenic in patients with chlamydial genital infections. Homology analyses suggested that YtgA is a soluble periplasmic protein and a component of an ATP-binding cassette (ABC) transport system for metals such as iron. Since little is known about iron transport in C. trachomatis, biochemical assays were used to determine the potential role of YtgA in iron acquisition. 59Fe binding and competition studies revealed that YtgA preferentially binds iron over nickel, zinc or manganese. Western blot and densitometry techniques showed that YtgA concentrations specifically increased 3–5-fold in C. trachomatis, when cultured under iron-starvation conditions rather than under general stress conditions, such as exposure to penicillin. Finally, immuno-transmission electron microscopy provided evidence that YtgA is more concentrated in C. trachomatis during iron restriction, supporting a possible role for YtgA as a component of an ABC transporter.

The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.

The poly(amino acid)s γ-poly(dl-glutamic acid) (gPGA) and ϵ-poly(l-lysine) (ePL) are known to be natural linear poly(amino acid)s secreted by Bacillus spp. and Streptomyces spp., respectively. In this study, a Streptomyces strain producing both ePL and gPGA was identified. Mass spectrometry and other analyses revealed that the gPGA is a mixture of oligomers consisting of 10–13 l-glutamic acid residues linked by isopeptide bonds. In contrast to the known Bacillus gPGA, the glutamic acid oligomers have a cyclodehydrated structure in each molecule. We previously reported that the ePL molecules secreted by the same Streptomyces strain disperse only slightly in an agar culture plate, as though they were larger molecules. This phenomenon is explicable by the observed polyion complex formation between the glutamic acid oligomers and ePLs. The glutamic acid oligomers control the ePL's dispersion, which would also affect the spatial distribution of the ePL's antimicrobial activity. Therefore, gene clustering or common use of the gene was presumed for biosynthesis of the two poly(amino acid)s. However, no gene for biosynthesis of the glutamic acid oligomer was found in the neighbouring region of that for ePL biosynthesis, and the glutamic acid oligomer was produced by a mutant in which the ePL biosynthetic gene was inactivated by gene disruption.

The hyperthermophilic archaeon Pyrococcus furiosus is a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely related Pyrococcus horikoshii and Pyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on the Pyr. furiosus genome. In the present study, these three genes were heterologously overexpressed in Escherichia coli to enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe–4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe–S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe–4S]-cluster-containing fumarate hydratases exemplified by MmcBC from Pelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase in Pyr. furiosus has yet to be established.

Trehalose accumulation is a common response to several stresses in the yeast Saccharomyces cerevisiae. This metabolite protects proteins and membrane lipids from structural damage and helps cells to maintain integrity. Based on genetic studies, degradation of trehalose has been proposed as a required mechanism for growth recovery after stress, and the neutral trehalase Nth1p as the unique degradative activity involved. Here we constructed a collection of mutants for several trehalose metabolism and transport genes and analysed their growth and trehalose mobilization profiles during experiments of saline stress recovery. The behaviour of the triple Δnth1Δnth2Δath1 and quadruple Δnth1Δnth2Δath1Δagt1 mutant strains in these experiments demonstrates the participation of the three known yeast trehalases Nth1p, Nth2p and Ath1p in the mobilization of intracellular trehalose during growth recovery after saline stress, rules out the participation of the Agt1p H+-disaccharide symporter, and allows us to propose the existence of additional new mechanisms for trehalose mobilization after saline stress.

The entomopathogenic fungus Beauveria bassiana is under intensive study as a pest biological control agent. B. bassiana produces several distinct single-cell types that include aerial conidia, in vitro blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, B. bassiana displays a dimorphic transition, producing in vivo specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of in vitro and in vivo insect haemolymph-derived B. bassiana cells. In vitro blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected Heliothis virescens haemolymph (in vivo cells) actively internalized FM4-64. The in vivo blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host–pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.