Dynamics of intracellular bacterial replication at the single cell level.

Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.

SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling.

Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates inside mammalian cells within membrane-bound compartments called Salmonella-containing vacuoles. Intracellular replication is dependent on the activities of several effector proteins translocated across the vacuolar membrane by the Salmonella pathogenicity island 2 (SPI-2)-type III secretion system (T3SS). This is accompanied by the formation in the vicinity of bacterial vacuoles of an F-actin meshwork, thought to be involved in maintaining the integrity of vacuolar membranes. In this study, we investigated the function of the SPI-2 T3SS effector SteC. An steC mutant strain was not defective for intracellular replication or attenuated for virulence in mice. However, the steC mutant was defective for SPI-2-dependent F-actin meshwork formation in host cells, although the vacuolar membranes surrounding mutant bacteria appeared to be normal. Expression of SteC in fibroblast cells following transfection caused extensive rearrangements of the F-actin cytoskeleton. Sequence analysis identified amino acid similarity between SteC and the human kinase Raf-1. A His-tagged SteC fusion protein had kinase activity in vitro and a point mutant lacking kinase activity was unable to induce F-actin rearrangements in vivo. We conclude that SPI-2-dependent F-actin meshwork formation depends on the kinase activity of SteC, which resembles more closely eukaryotic than prokaryotic kinases.

No contribution of angiotensin-converting enzyme (ACE) gene variants to severe obesity: a model for comprehensive case/control and quantitative cladistic analysis of ACE in human diseases.

Candidate gene analyses are often inconclusive owing to genetic or phenotypic heterogeneity, low statistical power, selection of nonfunctional SNPs, and inadequate statistical analysis of the genetic architecture. Angiotensin-converting enzyme (ACE) is involved in adipocyte growth and function and the ACE-processed angiotensin II inhibits adipocyte differentiation. Associations between body mass index (BMI) and ACE polymorphisms have been reported in general populations, but the contribution to severe obesity of this gene, which is located under an obesity genome-scan linkage peak on 17q23, is unknown. ACE is one of the most studied genes and markers responsible for variation in circulating ACE enzyme levels have been extensively characterised. Eight of these variants were genotyped in 1054 severely obese cases and 918 nonobese controls, as well as 116 nuclear families from the genome scan (n=447), enabling the known clades to be inferred. Qualitative analysis of individual single-nucleotide polymorphisms (SNPs), haplotypes, clades, and diploclades demonstrated no significant associations (P<0.05) after minimal correction for multiple testing. Quantitative analysis of clades and diploclades for BMI, waist-to-hip ratio, or ZBMI in children were also not significant. This rigorous, large-scale study of common, well-defined, severe polygenic obesity provides strong evidence that functionally relevant sequence variation in ACE, whether it is defined at the level of SNPs, haplotypes, or clades, is not associated with severe obesity in French Caucasians. Such a study design exemplifies the strategy needed to clearly define the contribution of the ACE gene to the plethora of complex genetic diseases where weak associations have been previously reported.

Association of melanin-concentrating hormone receptor 1 5' polymorphism with early-onset extreme obesity.

Murine models have been highly effective in identifying the monogenic forms of human obesity discovered to date. Melanin-concentrating hormone receptor 1 (MCHR1) has been shown to be significant in the downstream orexigenic activity of the leptin-melanocortin pathway by such models. In this study, the human MCHR1 gene was extensively characterized by sequencing 3.5 kb of coding, untranslated and intronic regions plus 1 kb of putative promoter region in 180 morbidly obese adults and 87 morbidly obese children, a total of >2.4 Mb of sequencing. Thirty-nine single nucleotide polymorphisms (SNPs) were found, seven of which encode an amino acid change. One mutation, R248Q, appeared to cosegregate with the obesity trait in one pedigree but was also found to be a rare polymorphism in control samples. To investigate the possible polygenic role of MCHR1, the six common SNPs (minor allele frequency >5%) found in the sequenced regions were then screened in 557 morbidly obese adults, 552 obese children, and 1,195 nonobese nondiabetic control subjects. The plausible promoter SNP, rs133068, was found to be associated with protection against obesity in obese children only (allele frequency P = 0.006 and genotype frequency P = 0.004). Most significant results were found when using a dominant model (P = 0.001, odds ratio 0.695 [95% CI 0.560-0.863]). However, similar associations were found when both adults and children were analyzed together (P = 0.006, 0.783 [0.658-0.930]), suggesting that severe forms of obesity with early onset may be associated with SNPs in MCHR1.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes.

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N( epsilon )-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression.

PURPOSE: Vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PlGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS: C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PlGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS: Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PlGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PlGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PlGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS: Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Localisation of WE-14 immunoreactivity in the developing mouse limbo-corneal nerve net.

WE-14 is generated in subpopulations of chromogranin A immunopositive endocrine cells and neurons including those innervating the anterior uvea. This study investigated WE-14 in intact sclero-limbo-corneal tissue from embryonic (E17), neonatal (N0-N16), and adult mice using immunocytochemistry and confocal scanning laser microscopy. Weak WE-14 immunostaining was observed at birth in nerve fibre tracts entering the corneal mid-stroma from the limbo-scleral junction. Immunopositive fibre tracts were evident throughout the cornea at N3; by N5 the mid-stromal plexus had begun to generate fibre populations extending toward the developing corneal epithelium, and some varicose fibres terminated amongst the developing epithelium. Immunostaining was evident at N7 in the developing limbo-scleral nerve net and some fibres exhibited a close association with unidentified vascular elements. By N11 and in subsequent neonates, the cornea had developed a distinct stratified nerve net composed of thick mid-stromal and thinner upper stromal nerve fibre bundles; both possessed populations of varicose WE-14 immunopositive fibres. In the adult, a sub-epithelial network of varicose WE-14 immunopositive fibres were evident at the limbo-scleral junction. Some fibres exhibited a close association with unidentified vascular elements, while others extended into the upper peripheral corneal stroma. WE-14 was evident in leashes throughout the basal corneal epithelium and generated fibres ramifying between the stratified epithelium with some fibres terminating amongst the outermost corneal epithelia. This study has demonstrated that WE-14 was evident in the limbo-corneal nerve net at birth and that its detection parallels corneal development to adulthood, where WE-14 is evident in a subpopulation of nerve fibres.

The Salmonella SPI-2 effector SseJ exhibits eukaryotic activator-dependent phospholipase A and glycerophospholipid : cholesterol acyltransferase activity.

Intracellular replication of Salmonella enterica serovar Typhimurium within membrane-bound compartments, called Salmonella-containing vacuoles, depends on the activities of several effector proteins translocated by the Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system. The SPI-2 effector protein SseJ shows similarity at the amino acid level to several GDSL lipases with glycerophospholipid : cholesterol acyltransferase (GCAT) activity. In this study, we show that catalytic serine-dependent phospholipase A (PLA) and GCAT activity of recombinant SseJ is potentiated by factor(s) present in HeLa cells, RAW macrophages and Saccharomyces cerevisiae. SseJ activity was enhanced with increasing amounts of, or preincubation with, eukaryotic cell extracts. Analysis of the activating factor(s) shows that it is soluble and heat- and protease-sensitive. We conclude that PLA and GCAT activities of SseJ are potentiated by proteinaceous eukaryotic factor(s).

SseL, a Salmonella deubiquitinase required for macrophage killing and virulence.

Expression of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system is controlled by the two-component regulatory system SsrA-SsrB. We used a transcriptomic approach to help define the SsrA-SsrB regulon. We identified a gene encoding an uncharacterized effector (SseL) whose translocation into host cells depends on the SPI-2 secretion system. SseL has similarities to cysteine proteases with deubiquitinating activity. A GST-SseL fusion protein specifically hydrolyzed mono- and polyubiquitin substrates in vitro with a preference for K63-linked ubiquitin chains. Ubiquitin-modified proteins accumulated in macrophages infected with Salmonella sseL mutant strains but to a lesser extent when infected with bacteria expressing active protein, demonstrating that SseL functions as a deubiquitinase in vivo. Salmonella sseL mutant strains did not show a replication defect or induce altered levels of cytokine production upon infection of macrophages but were defective for a delayed cytotoxic effect and were attenuated for virulence in mice.