Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production.

Clostridium difficile is responsible for significant mortality and morbidity in the hospitalized elderly. C. difficile spores are infectious and are a major factor contributing to nosocomial transmission. The Spo0A response regulator is the master regulator for sporulation initiation and can influence many other cellular processes. Using the ClosTron gene knockout system, we inactivated genes encoding Spo0A and a putative sporulation-associated sensor histidine kinase in C. difficile. Inactivation of spo0A resulted in an asporogeneous phenotype, whereas inactivation of the kinase reduced C. difficile sporulation capacity by 3.5-fold, suggesting that this kinase also has a role in sporulation initiation. Furthermore, inactivation of either spo0A or the kinase resulted in a marked defect in C. difficile toxin production. Therefore, Spo0A and the signaling pathway that modulates its activity appear to be involved in regulation of toxin synthesis in C. difficile. In addition, Spo0A was directly phosphorylated by a putative sporulation-associated kinase, supporting the hypothesis that sporulation initiation in C. difficile is controlled by a two-component signal transduction system rather than a multicomponent phosphorelay. The implications of these findings for C. difficile sporulation, virulence, and transmission are discussed.

Efficacy of hospital cleaning agents and germicides against epidemic Clostridium difficile strains.

OBJECTIVE: To compare the effects of hospital cleaning agents and germicides on the survival of epidemic Clostridium difficile strains. METHODS: We compared the activity of and effects of exposure to 5 cleaning agents and/or germicides (3 containing chlorine, 1 containing only detergent, and 1 containing hydrogen peroxide) on vegetative and spore forms of epidemic and non-epidemic C. difficile strains (3 of each). We carried out in vitro exposure experiments using a human fecal emulsion to mimic conditions found in situ. RESULTS: Cleaning agent and germicide exposure experiments yielded very different results for C. difficile vegetative cells, compared with those for spores. Working-strength concentrations of all of the agents inhibited the growth of C. difficile in culture. However, when used at recommended working concentrations, only chlorine-based germicides were able to inactivate C. difficile spores. C. difficile epidemic strains had a greater sporulation rate than nonepidemic strains. The mean sporulation rate, expressed as the proportion of a cell population that is in spore form, was 13% for all strains not exposed to any cleaning agent or germicide, and it was significantly increased by exposure to cleaning agents or germicides containing detergent alone (34%), a combination of detergent and hypochlorite (24%), or hydrogen peroxide (33%). By contrast, the mean sporulation rate did not change substantially after exposure to germicides containing either a combination of detergent and dichloroisocyanurate (9%) or dichloroisocyanurate alone (15%). CONCLUSIONS: These results highlight differences in the activity of cleaning agents and germicides against C. difficile spores and the potential for some of these products to promote sporulation.

Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon.

The last decade has witnessed extensive, and widespread, changes in scientific technologies that have impacted significantly upon the study of the life sciences. Arguably, the biggest advances in our comprehension of simple and complex biological processes have come as a consequence of obtaining the complete DNA sequence of organisms. It is likely that we will become accustomed to hearing of quantum leaps in the study and understanding of the biology of higher eukaryotes in the coming years, now that (near) complete genome sequences are available for man, mouse and rat. In this review, we will discuss the impact of genome sequence data, and the use of new scientific technologies that have emerged largely as consequence of the availability of this information, on the study of the master regulator of sporulation, Spo0A, in low G+C Gram-positive endospore-forming bacteria.

More is better: Lymph node harvesting in colorectal cancer.

INTRODUCTION: We sought to determine if lymph node harvesting and survival for CRC were comparable between laparoscopic and open resections in a community hospital setting. METHODS: A retrospective chart review of patients at two community hospitals who underwent open or laparoscopic resection for CRC between January 2008 and September 2013 was performed. RESULTS: Three hundred seventy-one patients had open and 110 had laparoscopic resections. There was no difference between open (17.85) and laparoscopic (18.91) approaches (p = 0.171) in the number of lymph nodes harvested. Patients who had more nodes removed tended toward improved survival, independent of stage (p = 0.052), an effect that was more pronounced in the open resection group (p = 0.031). There was no difference in survival between the open and laparoscopic groups overall (HR 1.52, p = 0.208). DISCUSSION: No survival advantage was found between the open and laparoscopic resection groups, affirming that the choice of operative approach for CRC does not affect the quality of the oncologic procedure in a community hospital setting. Patients who had more lymph nodes removed tended toward improved survival. The explanation for this effect remains unclear.

Virulence- and antibiotic resistance-associated two-component signal transduction systems of Gram-positive pathogenic bacteria as targets for antimicrobial therapy.

Two-component signal transduction systems are central elements of the virulence and antibiotic resistance responses of opportunistic bacterial pathogens. These systems allow the bacterium to sense and respond to signals emanating from the host environment and to modulate the repertoire of genes expressed to allow invasion and growth in the host. The integral role of two-component systems in virulence and antibiotic sensitivity, and the existence of essential two-component systems in several pathogenic bacteria, suggests that these systems may be novel targets for antimicrobial intervention. This review discusses the potential use of two-component systems as targets for antimicrobial therapy against Gram-positive pathogens and the current status in the development of inhibitors specific for these systems.

Two-component and phosphorelay signal-transduction systems as therapeutic targets.

Two-component and phosphorelay signal-transduction systems of pathogenic bacteria control the expression of genes encoding virulence factors and essential functions. Recent systematic gene inactivation studies have confirmed the integral role of two-component systems in the pathogenesis of diseases caused by several microorganisms and highlighted the validity of using these systems as targets for therapeutic intervention. Structural studies of signal-transduction proteins have recently revealed common features that may allow rational drug design for therapeutic intervention. In particular, the conserved domains of response regulators may represent the best targets for inhibition.

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review).

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.

Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiation of sporulation.

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and microESI-MS. NMR studies show that the divalent metals Ca(2+), Mg(2+) and Mn(2+) primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu(2+) show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. microESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu(2+), multiple metal-bound species are observed. Subsequent experiments reveal that Mg(2+) supports phosphotransfer between KinA and Spo0F, while Cu(2+) fails to support KinA phosphotransfer. Additionally, the presence of Cu(2+) at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.

Evolution of signalling in the sporulation phosphorelay.

Two-component and phosphorelay signal transduction systems are believed to function as environ-mental sensors that programme gene expression to the composition of the ecological niche in which a microbe normally resides. The question of how evolutionarily related bacteria that occupy different environments change their signal transduction pathways to adapt to such environments was asked of the sporulation phosphorelay of Bacillus subtilis, Bacillus halodurans, Bacillus anthracis and Bacillus stearothermophilus. Comparison of the primary amino acid sequence of phosphorelay proteins with the known structural and interactive properties of the B. subtilis proteins revealed that the amino acid residues of interaction surfaces between phosphorelay proteins and between a phosphorelay protein and DNA resist evolutionary change. The absolute conservation of interaction surfaces allowed the identification of sporulation sensor kinases in B. halodurans, B. anthracis and B. stearothermophilus. In these sensor kinases, the signal-sensing domains are vastly different in size and subdomain composition, with little apparent conservation between species, whereas the catalytic domains of these sensor kinases retain the high level of homology observed for the other phosphorelay proteins. Adaptation to new environments appears to result in rapid evolution of signalling domains to maximize environmental impact while maintaining identical protein-protein and protein-DNA contacts in the entire phosphorelay. In Clostridial genomes, only the Spo0A protein was found, suggesting that the anaerobic relatives of the Bacilli do not use a phosphorelay and phosphorylate Spo0A directly with sensor kinases.

Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system.

A series of chimeric alpha-amylase genes derived from amyL, which encodes the liquefying alpha-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active alpha-amylase was determined. Detectable alpha-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins.

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.