Erin C. Boyle

Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2 and SipA disrupt tight junction structure and function

Salmonella enterica serovar Typhimurium is a major cause of human gastroenteritis. Infection of epithelial monolayers by S. Typhimurium disrupts tight junctions that normally maintain the intestinal barrier and regulate cell polarity. Tight junction disruption is dependent upon the Salmonella pathogenicity island‐1 (SPI‐1) type 3 secretion system but the specific effectors involved have not been identified. In this study we demonstrate that SopB, SopE, SopE2 and SipA are the SPI‐1‐secreted effectors responsible for disruption of tight junction structure and function. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host‐derived products. Unlike wild‐type S. Typhimurium, ΔsopB, ΔsopE/E2, ΔsipA, or ΔsipA/sopB mutants, ΔsopB/E/E2 and ΔsipA/sopE/E2 mutants were unable to increase the permeability of polarized epithelial monolayers, did not disrupt the distribution or levels of ZO‐1 and occludin, and did not alter cell polarity. These data suggest that SPI‐1‐secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function.

Bacterial pathogenesis: exploiting cellular adherence

Cell adhesion molecules, such as integrins, cadherins, the immunoglobulin superfamily of cell adhesion molecules and selectins, play important structural roles and are involved in various signal transduction processes. As an initial step in the infectious process, many bacterial pathogens adhere to cell adhesion molecules as a means of exploiting the underlying signaling pathways, entering into host cells or establishing extracellular persistence. Often, bacteria are able to bind to cell adhesion molecules by mimicking or acting in place of host cell receptors or their ligands. Recent studies have contributed to our understanding of bacterial adherence mechanisms and the consequences of receptor engagement; they have also highlighted alternative functions of cell adhesion molecules.

Salmonella: from pathogenesis to therapeutics.

Although Salmonella enterica serovars are some of the best-studied bacterial pathogens, the field still has a long way to go, especially when one considers that (i) they cause significant morbidity and mortality worldwide; (ii) they have broad host ranges, but for unknown reasons, infections result in different diseases in different hosts; (iii) they are able to establish persistent infections, which serve as reservoirs for transmission/shedding; and (iv) they are increasingly resistant to many antibiotics. The Second Conference on Salmonella, sponsored by the American Society for Microbiology, was held in Victoria, Canada, from 9 to 13 September 2006. Over 200 participants from around the world gathered at the historic Empress Hotel to discuss their latest findings on Salmonella population biology, animal models, pathogenic mechanisms, clinical disease, and vaccination. The first highlight of the conference was the keynote address by Roy Curtiss III (Arizona State University, Temple). He embodied the spirit of the conference “from pathogenesis to therapeutics,” as his life with Salmonella has taken him from investigating bacterial genetics and pathogenic mechanisms to translating this research into therapeutic approaches and vaccine development. This review will highlight some of the central themes that arose during the course of the conference; however, as it is so brief, it will describe only a fraction of the many excellent talks and posters presented.

SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar Typhimurium

ABSTRACT Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. In S. enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors. Phenotypic analysis of ΔsseK1, ΔsseK2, and ΔsseK1/ΔsseK2 mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.

Salmonella Phage ST64B Encodes a Member of the SseK/NleB Effector Family

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.

Identification of cognate host targets and specific ubiquitylation sites on the Salmonella SPI-1 effector SopB/SigD

Salmonella enterica is a bacterial pathogen responsible for enteritis and typhoid fever. Virulence is linked to two Salmonella pathogenicity islands (SPI-1 and SPI-2) on the bacterial chromosome, each of which encodes a type III secretion system. While both the SPI-1 and SPI-2 systems secrete an array of effectors into the host, relatively few host proteins have been identified as targets for their effects. Here we use stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics to identify the host targets of the SPI-1 effector, SopB/SigD. The only host protein found to bind immunoprecipitated SopB was the small G-protein Cdc42. The interaction was confirmed by reciprocal immunoprecipitation, and Cdc42 also bound glutathione S-transferase-fused SopB and SopB delivered through infection by the bacteria, confirming the interaction by an orthogonal method and in a more physiological context. The region of SopB responsible for the interaction was mapped to residues 117-168, and SopB is ubiquitylated at both K19 and K541, likely as monoubiquitylation. SopB colocalizes with activated Cdc42 near the plasmalemma, but we found no evidence that SopB alone can alter Cdc42 activity. This approach is also widely applicable to identify binding partners to other bacterial effectors.

The impact of prenatal diagnosis on neural tube defect (NTD) pregnancy versus birth incidence in British Columbia.

The birth incidence of neural tube defect (NTD) cases in British Columbia (B.C.), and elsewhere in North America, is reported to be declining. This decline is being attributed to folic acid (FA) supplementation and food fortification, but 2nd trimester prenatal screening of pregnancies for NTDs and other congenital anomalies has increased during this timeframe, as well. This descriptive, population-based study evaluates the impact of prenatal screening of NTD-affected pregnancies on (1) pregnancy outcome and (2) reporting of NTD births to the provincial Health Status Registry (B.C.H.S.R.); and it assesses (3) the use of periconceptional FA supplementation. NTD cases were ascertained from medical records of health centres providing care to families with NTD-affected pregnancies and newborns; and from NTD cases reported to the B.C.H.S.R. In 1997–1999, the B.C.H.S.R. published a NTD incidence of 0.77/1000. In this study, 151 NTD-affected pregnancies were identified, with an incidence of 1.16/1000. Partial Reporting of induced abortions in a NTD incidence 45.5% low than the actual incidence. Medical records were available for review on 144/151 pregnancies. Prenatal screening identified 86.1% (124/144) of NTD-affected pregnancies, with 72.6% (90/124) resulting in pregnancy termination, and 27.4% (34/124) continuing to term. Use of FA supplementation in the periconceptional period was recorded in 36.4% of pregnancies (39/107). Thus in B.C. the decline in the NTD incidence is due predominantly to pregnancy terminations following prenatal diagnosis, which reduces the NTD incidence by 60%, from 1.16/1000 to 0.47/1000. Continued efforts for primary and the option of secondary prevention of NTDs are recommended in order to improve newborn health in B.C. and elsewhere. These interventions need to be monitored, however, for optimal health care planning.

Deception point: Peptidoglycan modification as a means of immune evasion

The ability to evade host immune surveillance is a critical virulence determinant for any pathogenic microorganism. The intracellular pathogen Listeria monocytogenes is highly adept at reaching privileged niches within the host without alerting immune responses. Upon infection, Listeria penetrates epithelial cells in the gastrointestinal tract and spreads silently from cell to cell. Once this barrier is breached, it has the ability to escape the phagosome and replicate within phagocytic cells, using them as vehicles to disseminate throughout the host (reviewed in ref. 1). The capacity to defend against the onslaught of immune defenses is another key aspect to being a successful pathogen. In this issue of PNAS, Boneca et al. (2) demonstrate that Listeria modifies peptidoglycan (PG) in its cell wall, thereby conferring resistance to the host bacteriolytic product, lysozyme. By preventing bacterial degradation and subsequent release of immunostimulants, PG modification is therefore also a means by which Listeria evades immune detection by host pattern–recognition receptors (PRRs). Importantly, although many pathogens are known to modify their PG, this report by Boneca et al. (2) is the first to demonstrate that PG N-deacetylation has an important role in virulence and evasion of host defense.

Treatment of infected lungs by ex vivo perfusion with high dose antibiotics and autotransplantation: A pilot study in pigs

The emergence of multi-drug resistant bacteria threatens to end the era of antibiotics. Drug resistant bacteria have evolved mechanisms to overcome antibiotics at therapeutic doses and further dose increases are not possible due to systemic toxicity. Here we present a pilot study of ex vivo lung perfusion (EVLP) with high dose antibiotic therapy followed by autotransplantation as a new therapy of last resort for otherwise incurable multidrug resistant lung infections. Severe Pseudomonas aeruginosa pneumonia was induced in the lower left lungs (LLL) of 18 Mini-Lewe pigs. Animals in the control group (n = 6) did not receive colistin. Animals in the conventional treatment group (n = 6) received intravenous application of 2 mg/kg body weight colistin daily. Animals in the EVLP group (n = 6) had their LLL explanted and perfused ex vivo with a perfusion solution containing 200 μg/ml colistin. After two hours of ex vivo treatment, autotransplantation of the LLL was performed. All animals were followed for 4 days following the initiation of treatment. In the control and conventional treatment groups, the infection-related mortality rate after five days was 66.7%. In the EVLP group, there was one infection-related mortality and one procedure-related mortality, for an overall mortality rate of 33.3%. Moreover, the clinical symptoms of infection were less severe in the EVLP group than the other groups. Ex vivo lung perfusion with very high dose antibiotics presents a new therapeutic option of last resort for otherwise incurable multidrug resistant pneumonia without toxic side effects on other organs.

Blood cytokine expression correlates with early multi-organ damage in a mouse model of moderate hypothermia with circulatory arrest using cardiopulmonary bypass

Cardiopulmonary bypass (CPB) with moderate hypothermic cardiac arrest (MHCA) is essential for prolonged complex procedures in cardiac surgery and is associated with postoperative complications. Although cytokine release provoked through MHCA under CPB plays a pivotal role in postoperative organ damage, the pathomechanisms are unclear. Here, we investigated the cytokine release pattern and histological organ damage after MHCA using a recently described mouse CPB model. Eight BALB/c mice underwent 60 minutes of circulatory arrest under CPB, were successively rewarmed and reperfused. Blood cytokine concentrations and liver and kidney function parameters were measured and histological changes to these organs were compared to control animals. Our results showed a marked increase in proinflammatory cytokines and histological changes in the kidney, lung, and liver after CPB. Furthermore, clinical chemistry showed signs of hemolysis and acute kidney injury. These results suggest early onset of solid organ injury which correlates with increased leukocyte infiltration. A better understanding of the interplay between pro-inflammatory cytokine activation and solid organ injury in this model of CBP with MHCA will inform strategies to reduce organ damage during cardiac surgeries in the clinic.