The role of productivity in the ecological and evolutionary dynamics of predator-prey interaction

Productivity is predicted to drive the ecological and evolutionary dynamics of predator-prey interaction through changes in resource allocation between different traits. However, resources are seldom constantly available and thus temporal variation in productivity could have considerable effect on the species' potential to evolve. To study this, three long-term microbial laboratory experiments were established where Serratia marcescens prey bacteria was exposed to predation of protist Tetrahymena thermophila in different prey resource environments. The consequences of prey resource availability for the ecological properties of the predator-prey system, such as trophic dynamics, stability, and virulence, were determined. The evolutionary changes in species traits and prey genetic diversity were measured. The prey defence evolved stronger in high productivity environment. Increased allocation to defence incurred cost in terms of reduced prey resource use ability, which probably constrained prey evolution by increasing the effect of resource competition. However, the magnitude of this trade-off diminished when measured in high resource concentrations. Predation selected for white, non-pigmented, highly defensive prey clones that produced predation resistant biofilm. The biofilm defence was also potentially accompanied with cytotoxicity for predators and could have been traded off with high motility. Evidence for the evolution of predators was also found in one experiment suggesting that co-evolutionary dynamics could affect the evolution and ecology of predator-prey interaction. Temporal variation in resource availability increased variation in predator densities leading to temporally fluctuating selection for prey defences and resource use ability. Temporal variation in resource availability was also able to constrain prey evolution when the allocation to defence incurred high cost. However, when the magnitude of prey trade-off was small and the resource turnover was periodically high, temporal variation facilitated the formation of predator resistant biofilm. The evolution of prey defence constrained the transfer of energy from basal to higher trophic levels, decreasing the strength of top-down regulation on prey community. Predation and temporal variation in productivity decreased the stability of populations and prey traits in general. However, predation-induced destabilization was less pronounced in the high productivity environment where the evolution of prey defence was stronger. In addition, evolution of prey defence weakened the environmental variation induced destabilization of predator population dynamics. Moreover, protozoan predation decreased the S. marcescens virulence in the insect host moth (Parasemia plantaginis) suggesting that species interactions outside the context of host-pathogen relationship could be important indirect drivers for the evolution of pathogenesis. This thesis demonstrates that rapid evolution can affect various ecological properties of predator-prey interaction. The effect of evolution on the ecological dynamics depended on the productivity of the environment, being most evident in the constant environments with high productivity.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Salmonella Typhimurium: Insight into the multi-faceted role of the LysR-type transcriptional regulators in Salmonella

The LysR-type transcriptional regulators (LTTRs) are widely distributed in various genera of prokaryotes LTTRs are DNA binding proteins that can positively or negatively regulate target gene expression and can also repress their own transcription Salmonella enterica comprises a group of Gram-negative bacteria capable of causing clinical syndromes that range from self-limiting diarrhoea to severe fibrinopurulent necrotizing enteritis and life threatening systemic disease. The survival and replication of Salmonella in macrophages and in infected host is brought about by the means of various two component regulatory systems, transporters and other virulence islands In Salmonella genome the existence of 44 LTTRs has been documented These LTTRs regulate bacterial stress response. systemic virulence in mice and also many virulence determinants in vitro. Here we focus on the findings that elucidate the structure and function of the LTTRs in Salmonella and discuss the importance of these LTTRs in making Salmonella a Successful pathogen...

Lactobacillus bacteremia, with special focus on the safety of probiotic Lactobacillus rhamnosus GG

Lactobacilli are gram positive rods, which belong to normal oropharyngeal, gastrointestinal and urogenital flora. They are widely used in food industry and as food additives. Although their virulence is presumed to be very low, opportunistic bacteremic infections, especially in immunocompromised hosts, have been detected. In the present study, the possible effects of increasing probiotic use of Lactobacillus rhamnosus GG (LGG) on the occurrence of bacteremia due to lactobacilli was evaluated on population level. In Finland, 90 Lactobacillus bacteremia cases were reported to the National Infectious Disease Register maintained by National Public Health Institute, during 1995-2000. Their proportion of all bacteremia cases was on average 0.24%, corresponding to 0.3 cases/100 000 inhabitants annually. In the Helsinki University Central Hospital district the corresponding proportion of all bacteremia cases was observed during 1990-2000. Despite LGG intake increased six folded no increasing trend in the occurrence of lactobacilli bacteremia was seen. A total of 85 Lactobacillus sp. blood isolates collected from different human bacteremic cases were characterised and compared with the commercial probiotic LGG strain. In species characterisation 46 L. rhamnosus strains, 12 L. fermentum and L. casei strains each, three each of L. gasseri, L. salivarius and L. jensenii species, two L. curvatus, and one each of L. plantarum, L. sakei, L. zeae and L. reuteri species were detected. Nearly half of the L. rhamnosus findings turned out to be indistinguishable from the probiotic LGG strain. Common predisposing factors to Lactobacillus bacteremia were immunosuppression, prior prolonged hospitalisation and prior surgical interventions. Severe or fatal comorbidities were found in 82% of the patients. Mortality at one month was 26% and severe underlying diseases were a significant predictor of death (OR 15.8). Antimicrobial susceptibility of Lactobacillus strains was species dependent. The Lactobacillus isolates were generally susceptible to imipenem, piperacillin-tazobactam, clindamycin and erythromycin, whereas all other than L. gasseri and L. jensenii species were not at all susceptible to vancomycin. The susceptibility to cephalosporin varied greatly even within species why they might not be recommended for treatment of Lactobacillus infections. The effect and safety of probiotic LGG preparation in amelioration of gastric symptoms and diarrhea in HIV-infected patients was evaluated. No significant differences in gastrointestinal symptoms or diarrhoea in LGG treated patients compared to placebo could be found. LGG was well tolerated with no adverse effects including bacteremic outbreaks could be observed. The use of probiotic LGG can be regarded safe in this immunocompromised patient group.

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Role of neutrophils in murine salmonellosis

Gastrointestinal infections with Salmonella enterica serovars have different clinical outcomes that range from localized inflammation to a life-threatening systemic disease in the case of typhoid fever. Using a mouse model of systemic salmonellosis, we investigated the contribution of neutrophils to the innate immune defense against Salmonella after oral infection. Neutrophil infiltration was dependent on the bacterial burden in various infected organs (Peyer's patches, mesenteric lymph nodes, spleen, and liver). However, the massive infiltration of neutrophils did not allow clearance of an infection with wild-type Salmonella, presumably due to protection of intracellular Salmonella against neutrophil activities. A Salmonella mutant strain deficient in Salmonella pathogenicity island 2 (SPI2) was able to infect systemic sites, but its replication was highly restricted and it did not cause detectable attraction of neutrophils. Neutrophil depletion by antibody treatment of mice did not restore the virulence of SPI2 or auxotrophic mutant strains, supporting the hypothesis that attenuation of the strains is not due to greater susceptibility to neutrophil killing. Our observations reveal that neutrophils have completely different roles during systemic salmonellosis and localized gastrointestinal infections. In the latter conditions, rapid neutrophil attraction efficiently prevents the spread of the pathogen, whereas the neutrophil influx is delayed during systemic infections and cannot protect against lethal bacteremia.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

Disease burden due to Streptococcus dysgalactiae subsp equisimilis (group G and C streptococcus) is higher than that due to Streptococcus pyogenes among Mumbai school children

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006-2008. GAS and GGS/GCS were identified on the basis of beta-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (1166/1504, 11%) was eightfold higher than the GAS carriage (22/1504, 1.5%) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.

Epidemiology, treatment and outcome of Staphylococcus aureus bacteremia and endocarditis

Staphylococcus aureus is the second most common bloodstream isolate both in community- and hospital-acquired bacteremias. The clinical course of S. aureus bacteremia (SAB) is determined by its complications, particularly by the development of deep infections and thromboembolic events. Despite the progress of antimicrobial therapy, SAB is still associated with high mortality. However, injection drug users (IDUs) tend to have fewer complications and better prognosis than nonaddicts, especially in endocarditis. The present study was undertaken to investigate epidemiology, treatment and outcome of S. aureus bacteremia and endocarditis in Finland. In particular, differences in bacterial strains and their virulence factors, and host immune responses were compared between IDUs and nonaddicts. In Finland, 5045 SAB cases during 1995-2001 were included using the National Infectious Disease Register maintained by National Public Health Institute. The annual incidence of SAB increased, especially in elderly. While the increase in incidence may partly be explained by better reporting, it most likely reflects a growing population at risk, affected by such factors as age and/or severe comorbidity. Nosocomial infections accounted for 51% of cases, with no change in their proportion during the study period. The 28-day mortality was 17% and remained unchanged over time. A total of 381 patients with SAB were randomized to receive either standard antibiotic treatment or levofloxacin added to standard treatment. Levofloxacin combination therapy did not decrease the mortality, lower the incidence of deep infections, nor did it speed up the recovery during 3 months follow-up. However, patients with a deep infection appeared to benefit from combination therapy with rifampicin, as suggested also by experimental data. Deep infections were found in 84% of SAB patients within one week after randomization, and they appeared to be more common than previously reported. Endocarditis was observed in 74 of 430 patients (17%) with SAB, of whom 20 were IDUs and 54 nonaddicts. Right-sided involvement was diagnosed in 60% of addicts whereas 93% of nonaddicts had left-sided endocarditis. Unexpectedly, IDUs showed extracardiac deep infections, thromboembolic events and severe sepsis with the same frequency as nonaddicts. The prognosis of endocarditis was better among addicts due to their younger age and lack of underlying diseases in agreement with earlier reports. In total, all 44 IDUs with SAB were included and 20 of them had endocarditis. An equal number of nonaddicts with SAB were chosen as group matched controls. Serological tests were not helpful in identifying patients with a deep infection. No individual S. aureus strain dominated in endocarditis among addicts. Characterization of the virulence factors of bacterial strains did not reveal any significant differences in IDUs and nonaddicts.