Use of chitin:DNA ratio to assess growth form in fungal cells.

BACKGROUND: Dimorphism, the ability to switch between a 'yeast-like' and a hyphal growth form, is an important feature of certain fungi, including important plant and human pathogens. The switch to hyphal growth is often associated with virulence, pathogenicity, biofilm formation and stress resistance. Thus, the ability to accurately and efficiently measure fungal growth form is key to research into these fungi, especially for discovery of potential drug targets. To date, fungal growth form has been assessed microscopically, a process that is both labour intensive and costly. RESULTS: Here, we unite quantification of the chitin in fungal cell walls and the DNA in nuclei to produce a methodology that allows fungal cell shape to be estimated by calculation of the ratio between cell wall quantity and number of nuclei present in a sample of fungus or infected host tissue. Using the wheat pathogen Zymoseptoria tritici as a test case, with confirmation in the distantly related Fusarium oxysporum, we demonstrate a close, linear relationship between the chitin:DNA ratio and the average polarity index (length/width) of fungal cells. We show the utility of the method for estimating growth form in infected wheat leaves, differentiating between the timing of germination in two different Z. tritici isolates using this ratio. We also show that the method is robust to the occurrence of thick-walled chlamydospores, which show a chitin:DNA ratio that is distinct from either 'yeast-like' blastospores or hyphae. CONCLUSIONS: The chitin:DNA ratio provides a simple methodology for determining fungal growth form in bulk tissue samples, reducing the need for labour-intensive microscopic studies requiring specific staining or GFP-tags to visualise the fungus within host tissues. It is applicable to a range of dimorphic fungi under various experimental conditions.

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes.

Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a host-specific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.

Molecular features of lipoprotein CD0873: A potential vaccine against the human pathogen Clostridioides difficile.

Clostridioides difficile is the primary cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis. The bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-negative strain. Immunization of mice with recombinant CD0873 prevented long-term gut colonization and was correlated with a strong secretory IgA immune response. We further present high-resolution crystal structures of CD0873, at 1.35-2.50 Å resolutions, offering a first view of the ligand-binding pocket of CD0873 and provide evidence that this lipoprotein adhesin is part of a tyrosine import system, an amino acid key in C. difficile infection. These findings suggest that CD0873 could serve as an effective component in a vaccine against C. difficile.

The Culture Environment Influences Both Gene Regulation and Phenotypic Heterogeneity in Escherichia coli.

Microorganisms shape the composition of the medium they are growing in, which in turn has profound consequences on the reprogramming of the population gene-expression profile. In this paper, we investigate the progressive changes in pH and sugar availability in the medium of a growing Escherichia coli (E. coli) culture. We show how these changes have an effect on both the cellular heterogeneity within the microbial community and the gene-expression profile of the microbial population. We measure the changes in gene-expression as E. coli moves from lag, to exponential, and finally into stationary phase. We found that pathways linked to the changes in the medium composition such as ribosomal, tricarboxylic acid cycle (TCA), transport, and metabolism pathways are strongly regulated during the different growth phases. In order to quantify the corresponding temporal changes in the population heterogeneity, we measure the fraction of E. coli persisters surviving different antibiotic treatments during the various phases of growth. We show that the composition of the medium in which ß-lactams or quinolones, but not aminoglycosides, are dissolved strongly affects the measured phenotypic heterogeneity within the culture. Our findings contribute to a better understanding on how the composition of the culture medium influences both the reprogramming in the population gene-expression and the emergence of phenotypic variants.

Trehalase plays a role in macrophage colonization and virulence of Burkholderia pseudomallei in insect and mammalian hosts.

Trehalose is a disaccharide formed from two glucose molecules. This sugar molecule can be isolated from a range of organisms including bacteria, fungi, plants and invertebrates. Trehalose has a variety of functions including a role as an energy storage molecule, a structural component of glycolipids and plays a role in the virulence of some microorganisms. There are many metabolic pathways that control the biosynthesis and degradation of trehalose in different organisms. The enzyme trehalase forms part of a pathway that converts trehalose into glucose. In this study we set out to investigate whether trehalase plays a role in both stress adaptation and virulence of Burkholderia pseudomallei. We show that a trehalase deletion mutant (treA) had increased tolerance to thermal stress and produced less biofilm than the wild type B. pseudomallei K96243 strain. We also show that the ΔtreA mutant has reduced ability to survive in macrophages and that it is attenuated in both Galleria mellonella (wax moth larvae) and a mouse infection model. This is the first report that trehalase is important for bacterial virulence.

Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation.

Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

Lovastatin interferes with the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts.

Statins have been shown to be cardioprotective; however, their interaction with endogenous cardioprotection by ischemic preconditioning and postconditioning is not known. In the present study, we examined if acute and chronic administration of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin affected the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts. Wistar rats were randomly assigned to the following three groups: 1) vehicle (1% methylcellulose per os for 12 days), 2) chronic lovastatin (15 mg.kg(-1).day(-1) per os for 12 days), and 3) acute lovastatin (1% methylcellulose per os for 12 days and 50 micromol/l lovastatin in the perfusate). Hearts isolated from the three groups were either subjected to a nonconditioning (aerobic perfusion followed by 30-min coronary occlusion and 120-min reperfusion, i.e., test ischemia-reperfusion), preconditioning (three intermittent periods of 5-min ischemia-reperfusion cycles before test ischemia-reperfusion), or postconditioning (six cycles of 10-s ischemia-reperfusion after test ischemia) perfusion protocol. Preconditioning and postconditioning significantly decreased infarct size in vehicle-treated hearts. However, preconditioning failed to decrease infarct size in acute lovastatin-treated hearts, but the effect of postconditioning remained unchanged. Chronic lovastatin treatment abolished postconditioning but not preconditioning; however, it decreased infarct size in the nonconditioned group. Myocardial levels of coenzyme Q9 were decreased in both acute and chronic lovastatin-treated rats. Western blot analysis revealed that both acute and chronic lovastatin treatment attenuated the phoshorylation of Akt; however, acute but not chronic lovastatin treatment increased the phosphorylation of p42 MAPK/ERK. We conclude that, although lovastatin may lead to cardioprotection, it interferes with the mechanisms of cardiac adaptation to ischemic stress.

Androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle.

Myostatin is an important negative regulator of skeletal muscle growth, while androgens are strong positive effectors. In order to investigate the possible interaction between myostatin and androgen pathways, we followed myostatin expression in the androgen-dependent levator ani (LA) muscle of the rat as a function of androgen status. By testosterone deprivation (castration), we induced LA growth arrest in young male rats, whilst atrophy in adult ones, however, both processes could be reversed by testosterone supplementation. After castration, a significant up-regulation of active myostatin protein (and its propeptide) was found, whereas the subsequent testosterone treatment reduced myostatin protein levels to normal values in both young and adult rats. Similarly, a testosterone-induced suppression of myostatin mRNA levels was observed in castrated adult but not in young animals. Altogether, androgens seem to have strong negative impact on myostatin expression, which might be a key factor in the weight regulation of LA muscle.