A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.     

Insulin resistance syndrome,body mass index and the risk of ischemic heart disease

Background

Many people who are not obese according to standard height and weight criteria may still display features of insulin resistance syndrome and thus be at high risk of ischemic heart disease. We sought to investigate the effect of cumulative features of insulin resistance syndrome on the risk of ischemic heart disease associated with variations in body mass index (BMI) among men who participated in the Québec Cardiovascular Study.

Methods

A cohort of 1824 nondiabetic men free of ischemic heart disease was evaluated at the 1985 baseline evaluation and followed for a period of 13 years, during which 284 first ischemic heart disease events were recorded. Relative hazards (RHs) of ischemic heart disease in 3 BMI groups (normal weight, overweight and obese) were estimated using Cox proportional hazards regression.

Results

Although obese men (BMI ≥ 30 kg/m²) were the most likely to accumulate features of insulin resistance syndrome, the univariate risk of ischemic heart disease in this group was not significantly increased compared with normal-weight men (BMI < 25 kg/m²) (RH 1.26, 95% confidence interval [CI] 0.88–1.80). However, obese men who accumulated more than 4 features of insulin resistance syndrome were at increased risk of ischemic heart disease (RH 1.81, 95% CI 1.02–3.19) compared with normal-weight men who had fewer than 3 features of the syndrome. Conversely, having more than 4 features of insulin resistance syndrome was associated with a 3-fold increase in the risk of ischemic heart disease among normal-weight men (RH 3.01, 95% CI 1.70–5.32).

Interpretation

Although obesity is an important risk factor for ischemic heart disease, variations in BMI alone poorly reflect the risk of ischemic heart disease associated with features of insulin resistance syndrome.In the late 1980s, Gerald Reaven proposed the concept of insulin resistance as the cornerstone of a plurimetabolic syndrome that included hypertriglyceridemia, low plasma high-density lipoprotein cholesterol concentrations, hypertension and hyperinsulinemia.¹ This syndrome has been referred to as syndrome X, insulin resistance syndrome and the metabolic syndrome.² Several other metabolic disturbances, such as an increase in the number of small, dense low-density lipoprotein particles, impaired fibrinolytic activity, a proinflammatory state, impaired postprandial lipoprotein metabolism and abdominal obesity, have been included as part of this syndrome over the years.^3,4Although obesity is an important component of insulin resistance syndrome, many people who are not obese according to standard height and weight criteria (i.e., body mass index [BMI]) may still display features of the syndrome. Indeed, studies have indicated that normal-weight subjects, whose BMI is less than 25 kg/m², may have as much as 40% fat in the abdominal area, a level that correlates closely with decreased insulin sensitivity⁵ and an atherogenic dyslipidemic state. In that context, the risk of ischemic heart disease among normal-weight people with insulin resistance syndrome and among obese people without insulin resistance syndrome represents a major gap in the existing literature. We therefore sought to investigate how features of insulin resistance syndrome affected the risk of ischemic heart disease associated with variations in BMI in a cohort of 1824 men who participated in the Québec Cardiovascular Study.     

Contribution of small mountainous rivers to particulate organic carbon input in the Bay of Biscay

The Nivelle River, a typical Pyrenean mountainous watershed reaching the Bay of Biscay (Atlantic Ocean), was sampled with high resolution during 1996. The particulate organic carbon (POC) contents during successive floods shows that there is a graduated impoverishment of the organic fraction of suspended particulate matter (SPM) from the first flood to the next ones, reaching a threshold value (3%) attributed to allochtonous fraction (soil). On the basis of the high frequency data of water discharge and POC concentration, an annual POC flux was established: 845 tons, corresponding to a specific POC flux of 5.3 tC km⁻² yr⁻¹. This value was obtained during a dry period and must be considered as a minimum value for longer time scale. The POC originated mostly from soil (55%) and riparian/litter (~40%) with a very minor (<5%) contribution of autochthonous POC. Thirty-two percent of the annual POC flux was carried in 1% of time and 66% in 10% of time. The specific POC yield, 5.3 tC km⁻² yr⁻¹, if extended to the whole mountainous area of the southern coast of the Bay of Biscay (19,000 km²), leads to an estimated POC flux around 100,000 t yr⁻¹. Although small Cantabrian mountainous rivers contributed to only 28% of the freshwater discharge in the Bay of Biscay, their POC load was estimated to account for 70% of the total POC inputs in the Bay.     

High-mobility group box 1 protein is an inflammatory mediator in necrotizing enterocolitis: protective effect of the macrophage deactivator semapimod

High-mobility group box 1 (HMGB1) is a late mediator of endotoxemia known to stimulate the production of proinflammatory cytokines that are putative mediators of intestinal inflammation associated with necrotizing enterocolitis (NEC). We hypothesized that HMGB1 is also involved in the pathogenesis of NEC. We examined the expression of HMGB1 and the effect of the novel drug semapimod on intestinal inflammation in an experimental model of NEC in neonatal rats. Newborn rats were subjected to hypoxia and fed a conventional formula by gavage (FFH) or were breast fed (BF). Rats were killed on day 4, and the distal ileum was harvested for morphological studies and Western blot analysis. FFH newborn rats but not BF controls developed intestinal inflammation similar to the histological changes observed in human NEC. We found that the expression of HMGB1 and its receptor for advanced glycation end products (RAGE) as well as that of other apoptosis/inflammation-related proteins (Bad, Bax, inducible nitric oxide synthase, and cyclooxygenase 2) was upregulated in the ileal mucosa of FFH newborn rats compared with BF animals. Administration of the drug semapimod inhibited the upregulation of those proteins and partially protected the animals against the FFH-induced intestinal injury. Elevated levels of HMGB1 were also found in ileal samples from infants undergoing intestinal resection for acute NEC. Our results implicate HMGB1 and RAGE as important mediators of enterocyte cell death and hypoxia-induced injury in NEC and support the hypothesis that inhibitors such as semapimod might play a therapeutic role in chronic intestinal inflammation characterized by this animal model.     

Carbon monoxide protects against the development of experimental necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of neonates that is increasing in incidence and often results in significant morbidity and mortality. Carbon monoxide (CO), a byproduct of the catabolism of heme, is known to have anti-inflammatory and antiapoptotic properties. In this study, we aimed to demonstrate that inhaled CO protects against the development of intestinal inflammation in a model of experimental NEC as well as decreases enterocyte cell death in vitro. Additionally, we also aimed to demonstrate that CO decreases enterocyte production of inducible nitric oxide synthase (iNOS) and nitric oxide (NO). Neonatal rats were exposed to intermittent hypoxia exposure and formula feeding to induce experimental NEC. Animals randomized to CO treatment were put in an environment containing 0.025% CO for 1 h/day on days 1-3 of life. All animals were killed on day 4 of life. In vitro experiments were performed with IEC-6 cells, a rat enterocyte cell line. Cells were examined for viability, iNOS production, and elaboration of NO. We found that CO diminished levels of serum inflammatory cytokines and nitrites, protected against intestinal inflammation, and decreased ileal iNOS production and protein nitration in a model of experimental NEC. In vitro, CO decreased cytokine- or hypoxia/endotoxin-induced iNOS and NO production. CO also abrogated TNF-alpha- and actinomycin D-induced apoptosis or hypoxia/endotoxin-induced cell death. In conclusion, 1 h of daily low-dose inhaled CO protected against the development of intestinal inflammation in a model of experimental NEC. iNOS and NO production were decreased by CO both in vivo and in vitro. CO may prove to be a useful clinical adjunct in the treatment of NEC.     

The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion

Host cell invasion in the Apicomplexa is unique in its dependency on a parasite actin-driven machinery and in the exclusion of most host cell membrane proteins during parasitophorous vacuole (PV) formation. This exclusion occurs at a junction between host cell and parasite plasma membranes that has been called the moving junction, a circumferential zone which forms at the apical tip of the parasite, moves backward and eventually pinches the PV from the host cell membrane. Despite having been described by electron microscopic studies 30 years ago, the molecular nature of this singular structure is still enigmatic. We have obtained a monoclonal antibody that recognizes the moving junction of invading tachyzoites of Toxoplasma gondii, in a pattern clearly distinct from those described so far for microneme and rhoptry proteins. The protein recognized by this antibody has been affinity purified. Mass spectrometry analysis showed that it is a rhoptry neck protein (RON4), a hypothetical protein with homologues restricted to Apicomplexa. Our findings reveals for the first time the participation of rhoptry neck proteins in moving junction formation and strongly suggest the conservation of this structure at the molecular level among Apicomplexa.     

Description of the data from the Collaborative Study on the Genetics of Alcoholism (COGA) and single-nucleotide polymorphism genotyping for Genetic Analysis Workshop 14

The data provided to the Genetic Analysis Workshop 14 (GAW 14) was the result of a collaboration among several different groups, catalyzed by Elizabeth Pugh from The Center for Inherited Disease Research (CIDR) and the organizers of GAW 14, Jean MacCluer and Laura Almasy. The DNA, phenotypic characterization, and microsatellite genomic survey were provided by the Collaborative Study on the Genetics of Alcoholism (COGA), a nine-site national collaboration funded by the National Institute of Alcohol and Alcoholism (NIAAA) and the National Institute of Drug Abuse (NIDA) with the overarching goal of identifying and characterizing genes that affect the susceptibility to develop alcohol dependence and related phenotypes. CIDR, Affymetrix, and Illumina provided single-nucleotide polymorphism genotyping of a large subset of the COGA subjects. This article briefly describes the dataset that was provided.     

A novel plasmid (pEMCJH03) isolated from moraxella catarrhalis possibly useful as a cloning and expression vector within this species

A preliminary screening study of six Moraxella catarrhalis isolates from primary school children in the Netherlands identified a small 3.5 kb plasmid (pEMCJH03), containing four open reading frames, which encoded three mobilizing and one replicase protein. Insertion of a kanamycin containing transposon (yielding pEMCJH04) allowed selection and isolation of the plasmid in Escherichia coli. Natural transformation of pEMCJH04 into M. catarrhalis was successful for 25% (3/12) of non-isogenic isolates, with no link between (lack of) transformability and genetic lineage or (lack of) transformability and complement phenotype, though the transformation efficiency was found to be rather low at approximately 615CFU/microg (range=60-1040CFU/microg ). This is only the second publication detailing a plasmid isolated from this important respiratory pathogen, and the ability to clone and express foreign proteins in M. catarrhalis using pEMCJH04 could help in the development of a vaccine expression vector, as well as providing a useful tool for studying promoter activity and in complementation studies of gene knockout mutants.