The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats.     

Bayesian classification and regression trees for predicting incidence of cryptosporidiosis

Background

Classification and regression tree (CART) models are tree-based exploratory data analysis methods which have been shown to be very useful in identifying and estimating complex hierarchical relationships in ecological and medical contexts. In this paper, a Bayesian CART model is described and applied to the problem of modelling the cryptosporidiosis infection in Queensland, Australia.

Methodology/Principal Findings

We compared the results of a Bayesian CART model with those obtained using a Bayesian spatial conditional autoregressive (CAR) model. Overall, the analyses indicated that the nature and magnitude of the effect estimates were similar for the two methods in this study, but the CART model more easily accommodated higher order interaction effects.

Conclusions/Significance

A Bayesian CART model for identification and estimation of the spatial distribution of disease risk is useful in monitoring and assessment of infectious diseases prevention and control.     

Factors associated with elevated ALT in an international HIV/HBV co-infected cohort on long-term HAART

Background

Previous studies have demonstrated that hepatitis B virus (HBV) infection increases the risk for ALT elevations in HIV-HBV co-infected patients during the first year of HAART; however, there is limited data on the prevalence of ALT elevations with prolonged HAART in this patient group.

Methods/Principal findings

To identify factors associated with ALT elevations in an HIV-HBV co-infected cohort receiving prolonged HAART, data from 143 co-infected patients on HAART enrolled in an international HIV-HBV co-infected cohort where ALT measurements were obtained every 6 months was analysed. A person-visit analysis was used to determine frequency of ALT elevation (≥2.5×ULN) at each visit. Factors associated with ALT elevation were determined using multivariate logistic regression with generalized estimating equations to account for correlated data. The median time on HAART at the end of follow-up was 5.6 years (range 0.4–13.3) years. During follow-up, median ALT was 36 U/L with 10.6% of person-visits classified as having ALT elevation. Most ALT elevations were grade 2 (86.5%), with only 13.5% of all ALT elevations grade 3 or higher. Univariate associations with ALT elevation (p<0.05) included history of AIDS, HBV DNA ≥2,000 IU/ml, HBeAg positive, study visit CD4 <200 cells/ml and nadir CD4 <200 cells/ml. In the multivariate analysis, only study visit CD4 <200 cells/ml (OR 2.07, 95%CI 1.04–4.11, p = 0.04) and HBeAg positive status (OR 2.22, 95%CI 1.03–4.79, p = 0.04) were independently associated with ALT elevation.

Conclusions

In this HIV-HBV co-infected cohort, elevated ALT after >1 year of HAART was uncommon, and severe ALT elevations were rare. HIV-HBV co-infected patients on long-term HAART who are either HBeAg positive or have a CD4 count of <200 cells/ml are at increased risk for ALT elevations.     

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: a microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.     

RNA interference: past, present and future

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs) which are produced from long double-stranded RNAs by RNAse II-like enzyme Dicer. The resulting siRNAs are incorporated into a RNA-induced silencing complex (RISC) that targets and cleaves mRNA complementary to the siRNAs. Since its inception in 1998, RNAi has been demonstrated in organisms ranging from trypanosomes to nematodes to vertebrates. Potential uses already in progress include the examination of specific gene function in living systems, the development of anti-viral and anti-cancer therapies, and genome-wide screens. In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.     

Characterization of Salmonella enterica subspecies I genovars by use of microarrays

Subspecies 1 of Salmonella enterica is responsible for almost all Salmonella infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most Salmonella infections in humans and domestic animals. The gene contents of 79 strains from the most prevalent serovars were profiled by microarray analysis. Strains within the same serovar often differed by the presence and absence of hundreds of genes. Gene contents sometimes differed more within a serovar than between serovars. Groups of strains that share a distinct profile of gene content can be referred to as "genovars" to distinguish them from serovars. Several misassignments within the Salmonella reference B collection were detected by genovar typing and were subsequently confirmed serologically. Just as serology has proved useful for understanding the host range and pathogenic manifestations of Salmonella, genovars are likely to further define previously unrecognized specific features of Salmonella infections.     

ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1

We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well defined standard growth condition of Luria-Bertani medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, the two-component regulatory genes hilC/hilD, sirA/barA, and ompR, which are known to modulate expression from the hilA promoter (hilAp) under so-called "inducing conditions" (Luria-Bertani medium containing 0.3 m NaCl without aeration), acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by the addition of serine hydroxamate did not induce hilAp in a RelA(+) SpoT(+) strain suggested that, in addition to ppGpp, some other alteration accompanying entry into the stationary phase might be necessary for induction. It is speculated that during the course of infection Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, which allows expression of Salmonella virulence genes, including SPI1 genes.     

Nonuniformity of axial and circumferential remodeling of large coronary veins in response to ligation

The pressure-induced remodeling of coronary veins is important in coronary venous retroperfusion. Our hypothesis is that the response of the large coronary veins to pressure overload will depend on the degree of myocardial support. Eleven normal Yorkshire swine from either sex, weighing 31-39 kg, were studied. Five pigs underwent ligation of the left anterior descending (LAD) vein, and six served as sham-operated controls. The ligation of the coronary vein caused an increase in pressure intermediate to arterial and venous values. After 2 wk of ligation, the animals were euthanized and the coronary vessels were perfusion-fixed with glutaraldehyde. The LAD vein was sectioned, and detailed morphometric measurements were made along its length from the point of ligation near the base down to the apex of the heart. The structural remodeling of the vein was circumferentially nonuniform because the vein is partially embedded in the myocardium; it was also axially nonuniform because it is tethered to the myocardium to different degrees along its axial length. The wall area was significantly larger in the experimental group, whereas luminal area in the proximal LAD vein was significantly smaller in the same group compared with sham-operated controls. The wall thickness-to-radius ratio was also significantly larger in the experimental group in proportion to the increase in pressure. The major conclusion of this study is that the response of the vein depends on the local wall stress, which is, in part, determined by the surrounding tissue. Furthermore, the geometric remodeling of the coronary vein restores the circumferential stress to the homeostatic value.