The YrbE phospholipid transporter of Salmonella enterica serovar Typhi regulates the expression of flagellin and influences motility,adhesion and induction of epithelial inflammatory responses

ABSTRACT

Salmonella enterica

serovar Typhi is the etiologic agent of typhoid fever, a major public health problem in the developing world. Moving toward and adhering to the intestinal epithelium represents key initial steps of infection by S. Typhi. We examined the role of the S. Typhi yrbE gene, which encodes an inner membrane phospholipid transporter, in these interactions with epithelial cells. Disruption of yrbE resulted in elevated expression of flagellin and a hypermotile phenotype. It also significantly reduced the ability of S. Typhi to adhere to the HeLa epithelial cell line and to polarized primary epithelial cells derived from human ileal organoids. Interestingly, the yrbE-deficient strain of S. Typhi induced higher production of interleukin-8 from the primary human ileal epithelial cell monolayers compared to the wild-type bacteria. Deletion of the flagellin gene (fliC) in the yrbE-deficient S. Typhi inhibited motility and attenuated interleukin-8 production, but it did not correct the defect in adhesion. We also disrupted yrbE in S. Typhimurium. In contrast to the results in S. Typhi, the deficiency of yrbE in S. Typhimurium had no significant effect on flagellin expression, motility or adhesion to HeLa cells. Correspondingly, the lack of yrbE also had no effect on association with the intestine or the severity of intestinal inflammation in the mouse model of S. Typhimurium infection. Thus, our results point to an important and serovar-specific role played by yrbE in the early stages of intestinal infection by S. Typhi.     

Isolation and Characterisation of Bacteriophages with Activity against Invasive Non-Typhoidal Salmonella Causing Bloodstream Infection in Malawi

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.     

Severe oral infection due to <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> during induction chemotherapy for acute myeloid leukemia

We report a case of severe oral infection with a high fever due to Lactobacillus rhamnosus during induction chemotherapy for acute myeloid leukemia. The patient did not improve on treatment with meropenem, clindamycin, or vancomycin until neutrophil recovery. Since L. rhamnosus GG is used in dairy products, and the patient ingested dairy products daily before starting chemotherapy, we suspected an association between the ingestion of dairy products and the development of infection. Pulsed-field gel electrophoresis using two different restriction enzymes showed that the strain isolated from the patient was identical to the L. rhamnosus GG strain isolated from dairy products and ATCC #53103. This was confirmed by a PCR assay with species-specific L. rhamnosus GG primers. Since Lactobacillus infection, particularly L. rhamnosus infection, can be fatal in immunocompromised hosts, we should consider Lactobacillus as a causative organism when Gram-positive rods are detected during treatment with broad-spectrum antibiotics and vancomycin. The causal association between the ingestion of dairy products containing Lactobacillus and Lactobacillus infection in immunocompromised hosts warrants further study.     

Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to <Emphasis Type="Italic">Salmonella</Emphasis> spp. with reduced fluoroquinolone susceptibility: a case series

Background

Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases.

Methods

Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed.

Results

Seven of 21 (33%) S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L).Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization.

Conclusions

Our cases provide further evidence that reduced fluoroquinolone susceptibility of S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extra-intestinal Salmonella spp. for reduced fluoroquinolone susceptibility.

     

Impact of a probiotic fermented milk in the gut ecosystem and in the systemic immunity using a non-severe protein-energy-malnutrition model in mice

Background

Malnutrition affects the immune response, causing a decrease of defence mechanisms and making the host more susceptible to infections. Probiotics can reconstitute the intestinal mucosa and stimulate local and systemic immunity. The aim of this work was evaluate the effects of a probiotic fermented milk as a complement of a re-nutrition diet, on the recovery of the intestinal barrier, and mucosal and systemic immune functions in a murine model of non-severe protein-energy-malnutrition. Its potential protection against Salmonella enterica serovar Typhimurium (S. Typhimurium) infection was also analyzed.

Methods

Mice were undernourished and divided into 3 groups according to the dietary supplement received during re-nutrition (milk, probiotic fermented milk or its bacterial free supernatant) and compared to well-nourished and malnourished mice. They were sacrificed previous to the re-nutrition and 5 days post re-nutrition. The phagocytic activity of macrophages from spleen and peritoneum and the changes in the intestinal histology and microbiota were evaluated. Different immune cell populations and cytokine productions were analyzed in the small intestine tissues. The effect of the re-nutrition supplements on the systemic immunity using OVA antigen and against an infection with S. Typhimurium was also studied.

Results

Probiotic fermented milk was the most effective re-nutrition diet that improved the intestinal microbiota. Its administration also increased the number of IgA+ cells, macrophages and dendritic cells. The production of different cytokine (IFN-γ, TNF-α, IL-12) by these cells and the phagocytic activity in peritoneum and spleen was also increased. This re-nutrition diet also stimulated the systemic immune response against OVA antigen which was diminished after the malnutrition period and also improved the host response against S. Typhimurium, decreasing the spread of pathogenic bacteria to the liver and the spleen. The importance of the metabolites released during milk fermentation was also demonstrated through the analysis of the bacterial free supernatant obtained from the probiotic fermented milk, but the whole product showed the best effects in the parameters evaluated in this study.

Conclusions

The administration of probiotic fermented milk as a dietary supplement during the re-nutrition process in a murine immunodeficiency model by malnutrition could be a good adjuvant diet to improve the gut and systemic immune response for the protection against Salmonella infection.     

Increasing prevalence and dissemination of invasive nontyphoidal Salmonella serotype Typhimurium with multidrug resistance in hospitalized patients from southern Brazil

Introduction

Nontyphoidal Salmonella serotypes are the main cause of human food-borne infection, including several hospitalization cases in the developing countries.

Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils

Background/Aims

The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods

Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results

Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions

Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.     

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

The innate immune system senses microbial pathogens through recognition of conserved entities collectively referred to as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). These entities interact with conserved pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and C-type lectin receptors (CLRs) that are expressed by immune cells and other cell types. Activation of PRRs by PAMPs is dictated by the availability and expression levels of PAMPs at different stages of infection and results in host responses which are vital for inflammation and immunity against pathogens (1). However, some pathogens, including Salmonella spp., a facultative intracellular pathogen, have evolved the ability to use these host responses for their own replication and establishment of infection (2).Flagellin, the monomeric protein constituting bacterial flagella, is one of the key Salmonella effector molecules which binds and activates membrane-bound TLR-5 as well as the cytosolic sensor NLRC4 and plays a major role in generating inflammatory responses (3–5). In macrophages, flagellin as well as the rod protein PrgJ, which are inadvertently released into the host cytosol by the type III secretion system (T3SS), are detected by the NAIPs. In mice, seven NAIPs are present of which NAIP1 senses the T3SS needle protein, NAIP2 detects the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin (6–9). Humans however encode a single functional NAIP which has been recently shown to broadly detect multiple T3SS proteins and flagellin (10). Ligand binding to the NAIPs leads to recruitment and oligomerization of NLRC4 (11, 12). Activation of the NAIP-NLRC4 inflammasome by these effectors and activation of the NLRP3 inflammasome by an as yet unidentified aconitase-regulated Salmonella effector (13–15) results in caspase-1-dependent pyroptosis and production of active IL-1β which promotes clearance of the bacterium and protects the host against Salmonella (13, 16, 17). It is believed that as infection progresses, Salmonella circumvents this host-protective response by suppressing the expression of flagellin to lower than the resting levels usually expressed by bacteria in culture (18). Down-regulation of flagellin is essential for the bacterium to establish successful infection. Previous work has shown that a Salmonella Typhimurium strain modified to constitutively express flagellin (ST-FliC^ON) and therefore unable to naturally down-regulate flagellin expression is avirulent and cleared successfully from the host compared to its wild-type (WT) counterpart (17). Despite this central role of flagellin in Salmonella pathogenesis, the molecular mechanisms that regulate the physiological switch of Salmonella from a flagellin-high to a flagellin-low phenotype and aid in establishment of an intracellular niche within macrophages in vivo are incompletely understood.Upon entry into macrophages, Salmonella resides in a vacuole called the Salmonella-containing vacuole (SCV) where it shuts down expression of the Salmonella pathogenicity island 1 (SPI-1) and concomitantly switches on expression of Salmonella pathogenicity island 2 (SPI-2), which is activated by the PhoP/PhoQ two-component system (19) and encodes genes required for intracellular replication. Prior work has shown that shutdown of SPI-1 in growth media that mimic conditions associated with the SCV such as acidic pH and low Mg²⁺ is also accompanied by repression of flagellin (20, 21). This is because low pH and low Mg²⁺ activate the PhoP/PhoQ system (20, 22, 23) and activated PhoP is believed to suppress expression of flagellin (21). A noteworthy issue relating to these early studies is that effects on PhoP/PhoQ-regulated genes were examined only during in vitro culture of bacteria in growth medium and not in the context of S. Typhimurium residence within macrophages. Therefore, the physiological contribution of these mechanisms to flagellin repression of intracellular Salmonella remains debatable. For example, contrary studies have shown that the effect of low pH on flagellin protein expression is observed only at a very low pH (pH = 3) and not at pH 5 (20) which is close to the physiologically relevant pH of the SCV (24, 25). Likewise, neither variation of extracellular Mg²⁺ nor reduced Mg²⁺ in the SCV was found to play a role in PhoP activation by Salmonella inside macrophages (26). Consequently, the regulatory mechanisms conventionally thought to repress flagellin expression by Salmonella remain controversial and there is scarce evidence to suggest that these factors are responsible for down-regulation of flagellin by bacteria residing within macrophages. Moreover, the physiological mechanisms that regulate repression of flagellin in vivo are unknown.In this study we describe a host innate immune circuit that regulates expression of Salmonella flagellin during both the early/extracellular and the later/intracellular phases of macrophage infection with this pathogen. We find that during early infection of macrophages with S. Typhimurium, rapid NLRC4 inflammasome-dependent macrophage pyroptosis is necessary and sufficient for releasing a host lysophospholipid stimulus that promotes synthesis and release of flagellin from Salmonella. Unexpectedly, these host factors regulate not only the initial increase in flagellin production but also the later down-regulation of flagellin by Salmonella inside macrophages. This later effect is mediated by a natural type 1 IFN-dependent host negative feedback response that represses expression of NLRC4 and the lysophospholipid biosynthetic enzyme calcium-independent phospholipase A2 (iPLA2) within cells, causing a decline in intracellular lysophospholipids over time, which promotes eventual down-regulation of flagellin by intracellular bacteria. Our data identify host NLRC4 inflammasome activity as a temporal and biphasic regulator of expression of its own bacterial ligand, flagellin. We also describe a physiologically relevant type 1 IFN-mediated host mechanism that controls switching of Salmonella from a flagellin-high to a flagellin-low phenotype within macrophages in vivo. These findings have important implications for understanding the intricate evolutionary adaptations that shape host–pathogen cross-talk.     

H. pylori Infection Is Associated with DNA Damage of Lgr5-Positive Epithelial Stem Cells in the Stomach of Patients with Gastric Cancer

Background

H. pylori (Hp) infection is a major risk factor in gastric carcinogenesis leading to epithelial mutagenesis, and may affect gastric epithelial stem cells.

Aims

To characterize the expression of Lgr5, a marker of epithelial stem cells in human gastric mucosa, to determine whether Hp infection affects Lgr5-positive epithelial cells (LPECs) and whether LPECs are susceptible to DNA damage associated with Hp infection.

Methods

Lgr5 expression was characterized in non-neoplastic gastric mucosa from 52 patients (34 with and 18 without gastric cancer (GC); 21 Hp-positive (Hp+) and 31 Hp-negative (Hp?)) by immunohistochemical and immunofluorescence staining. To determine the extent of DNA damage in LPECs, nuclear 8-hydroxydeoxyguanosine (8OHdG), a marker of DNA damage associated with oxidative stress, was measured by quantitative spectral image analysis.

Results

LPECs were primarily present in gastric antrum. Higher numbers of LPECs were seen in Hp+ than in Hp? non-neoplastic mucosa of GC patients, P = .006, but not in patients without GC. 8OHdG levels in LPECs were significantly higher than in Lgr5-negative epithelial cells in Hp+ GC patients (P = .012) but not in Hp? cases (P = .414), whereas no difference was seen between Hp+ and Hp? mucosa of patients without GC.

Conclusions

The Lgr5-positive epithelial stem cell pool is expanded in Hp-associated gastritis in the antrum of patients with GC. In GC patients with active Hp infection, LPECs may be more susceptible to DNA damage than Lgr5-negative epithelial cells, suggesting that Hp infection may contribute to GC risk by affecting epithelial stem cells in the human stomach.     

Comparison of sporadic cases of Salmonella Typhimurium with other Salmonella serotypes in Castellon (Spain): case-case study

Introduction

Salmonella infections (SI) are common in Spain. The aim of this study was to appraise risk factors and the clinical characteristics of sporadic Salmonella Typhimurium infections compared with other sporadic salmonella serotype infections (OSI).

Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses

Background

Many Gram-positive and Gram-negative bacteria produce large quantities of indole as an intercellular signal in microbial communities. Indole demonstrated to affect gene expression in Escherichia coli as an intra-species signaling molecule. In contrast to E. coli, Salmonella does not produce indole because it does not harbor tnaA, which encodes the enzyme responsible for tryptophan metabolism. Our previous study demonstrated that E. coli-conditioned medium and indole induce expression of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium for inter-species communication; however, the global effect of indole on genes in Salmonella remains unknown.

Results

To understand the complete picture of genes regulated by indole, we performed DNA microarray analysis of genes in the S. enterica serovar Typhimurium strain ATCC 14028s affected by indole. Predicted Salmonella phenotypes affected by indole based on the microarray data were also examined in this study. Indole induced expression of genes related to efflux-mediated multidrug resistance, including ramA and acrAB, and repressed those related to host cell invasion encoded in the Salmonella pathogenicity island 1, and flagella production. Reduction of invasive activity and motility of Salmonella by indole was also observed phenotypically.

Conclusion

Our results suggest that indole is an important signaling molecule for inter-species communication to control drug resistance and virulence of S. enterica.     

Chemoprevention by Probiotics During 1,2-Dimethylhydrazine-Induced Colon Carcinogenesis in Rats

Background

Probiotics are believed to have properties that lower the risk of colon cancer. However, the mechanisms by which they exert their beneficial effects are relatively unknown.

Aim

To assess the impact of probiotics in preventing induction of colon carcinogenesis in rats.

Methods

The rats were divided into six groups viz., normal control, Lactobacillus plantarum (AdF10)-treated, Lactobacillus rhamnosus GG (LGG)-treated, 1,2-dimethylhydrazine (DMH)-treated, L. plantarum (AdF10) + DMH-treated and L. rhamnosus GG (LGG) + DMH-treated. Both the probiotics were supplemented daily at a dose of 2 × 10¹⁰ cells per day. DMH at a dose of 30 mg/kg body weight was administered subcutaneously twice a week for the first 4 weeks and then once every week for a duration of 16 weeks. Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and catalase as protein expression of genes involved in apoptosis were assessed during DMH-induced colon carcinogenesis in rats.

Results

DMH treatment decreased the activity of GSH, GPx, GST, SOD and catalase. However, AdF10 and LGG supplementation to DMH-treated rats significantly increased the activity of these enzymes. Further, DMH treatment revealed alterations in the protein expressions of various genes involved in the p53-mediated apoptotic pathway such as p53, p21, Bcl-2, Bax, caspase-9 and caspase-3, which, however, were shifted towards normal control levels upon simultaneous supplementation with probiotics.

Conclusion

The present study suggests that probiotics can provide protection against oxidative stress and apoptotic-related protein disregulation during experimentally induced colon carcinogenesis.

     

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.A critical step in disease pathogenesis for many clinically important bacteria is their ability to infect and survive within host cells such as macrophages. Salmonella enterica, a pathogen that resides and replicates within macrophages, causes a range of life-threatening diseases in humans and animals, and accounts for 28 million cases of enteric fever worldwide each year (1). S. enterica infects phagocytes by a process that requires cytoskeletal reorganization (2). This bacterium resides in a Salmonella-containing vacuole (SCV) within host macrophages, and this intracellular lifestyle enables them to avoid extracellular antimicrobial killing, evade adaptive immune responses, and potentially to spread to new sites to seed new infectious foci within host tissue, which eventually develop into granulomas (3). Survival and growth of S. enterica within phagocytes is critical for virulence (4) and host restriction of the intracellular bacterial load is, therefore, paramount in surviving salmonellosis. Salmonella delivers microbial effector proteins into the host cell via the type III secretion systems (T3SS), mediated by the Salmonella pathogenicity island-1 and -2 (SPI-1 and SPI-2), to subvert cellular functions and facilitate intracellular survival (5).Microbes are recognized by macrophages through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), which initiate innate immune responses, including cytokine production and pathogen killing (6). NLRs drive the formation of inflammasomes—macromolecular protein complexes—comprising one or more NLRs, usually an adaptor protein (ASC) and the effector protein caspase-1, which then cleaves prointerleukin-1β (IL-1β) and pro–IL-18 into biologically active cytokines, and initiates macrophage cell death by pyroptosis (7). NLRC4, in concert with NAIPs 1, 2, 5, and 6, is a key PRR that forms an inflammasome complex upon sensing flagellin and/or the inner rod or needle proteins (PrgJ and PrgI, respectively) of the SPI-1 T3SS of S. enterica serovar Typhimurium (S. Typhimurium) (8–11). Activation of the NLRC4 inflammasome by Salmonella infection results in IL-1β and IL-18 production driven by an ASC-dependent pathway and macrophage pyroptosis driven by an ASC-independent pathway (12, 13). A second, noncanonical, NLR signaling pathway has been described, which requires caspase-11 to initiate delayed cell death and NLRP3 inflammasome activation (14–16). Effective clearance of Salmonella infection in host cells may therefore require a coordinated effort between different inflammasome signaling pathways.We, and others, have shown that NLRC4 is important in regulating bacterial burden of S. Typhimurium in vivo (17–19). A recent study revealed that Salmonella-infected epithelial cells are extruded from the intestinal epithelium in a process that requires NLRC4 (20). The molecular mechanism behind how NLRC4 restricts bacterial burden in macrophages infected with Salmonella is still unknown. Here, we identify an actin-dependent mechanism that controls NLRC4-mediated regulation of bacterial replication in macrophages infected with S. Typhimurium. Activation of NLRC4 in infected macrophages mediates the production of reactive oxygen species (ROS) to inhibit bacterial replication and limits additional bacterial uptake by inducing mechanical stiffening the cell via actin polymerization. Overall, we describe a previously unidentified effector mechanism, governed by actin and the NLRC4 inflammasome, to control Salmonella infection in macrophages.     

Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells

Background

Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. fluorescens (biovar 1) are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of P. fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of P. fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen P. aeruginosa PAO1.

Results

Both strains of P. fluorescens were found to decrease the transepithelial resistance (TER) of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers.

Conclusions

The present work shows for the first time, that P. fluorescens is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two P. fluorescens strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the P. fluorescens specific I2 antigen in the serum of patients with Crohn's disease.     

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.     

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Salmonella enterica is a gram-negative, facultative intracellular human pathogen, annually causing more than 100 million food- and waterborne infections worldwide. Salmonella Typhimurium causes severe gastroenteritis, which could turn into a systemic infection in children, immune-compromised, or elderly people (1, 2). Concurrently, multidrug resistant bacteria are globally emerging and threatening our health systems, calling for a better understanding of the underlying virulence mechanism and host response.Pathogenic bacteria have evolved the inherent ability to infect and to establish their niche within host cells. For colonizing nonphagocytic cells such as epithelial cells, Salmonella uses a trigger mechanism–based entry mode. Bacterial virulence factors are then injected via a Type III-secretion system (T3SS) into the host cell to induce cytoskeletal rearrangements leading to membrane ruffling and macropinocytosis-driven internalization into a sealed phagosome (3, 4). This specialized compartment is referred to as the Salmonella-containing vacuole (SCV) and serves as the intracellular replicative niche by hiding the bacteria from the humoral and cell-autonomous immune response (5). Salmonella invasion requires a cooperative action of several bacterial effector proteins hijacking multiple host targets. One of the main targets forcing Salmonella´s uptake is the actin cytoskeleton by subverting the host Rho GTPases system. Bacterial effector proteins such as SopE/SopE2 mimic host nucleotide exchange factors (GEFs) to stimulate Rac1 and CDC42 activity (6, 7). Once GTP-activated, Rho GTPases stimulate downstream pathways to drive actin filament (F-actin) assembly and rearrangement.The actin cytoskeleton network is regulated by actin-binding proteins (ABPs), which orchestrate assembly and disassembly of actin in higher networks (8). Monomeric, globular actin (G-actin) is nucleated into new actin filaments, or the existing F-actin is elongated, stabilized, or disassembled by ABPs. The major actin nucleation factor is the multimeric Arp2/3 complex, which generates branched actin filament networks. Formins generate long unbranched actin filaments and represent another actin nucleation family. Together with actin nucleation-promoting factors, small Rho GTPases control ABPs in a spatiotemporal manner. Actin polymerization and membrane ruffling are necessary for Salmonella invasion. Following Salmonella internalization, the SCV undergoes SPI-1–dependent biogenesis and is transported to a juxtanuclear position at 1 to 2 h postinfection (pi). At later time-points (4 to 6 h pi), SPI-2–dependent effector proteins are expressed to further mature the SCV, allowing bacterial proliferation. Pioneering work described that, at later stages of the infection (≥6 h pi), an actin meshwork around the SCV stabilizes and protects the vacuolar niche (9–13).Here, we report SIK2 as a Salmonella resistance factor and a regulator of the actin cytoskeleton. SIK2 belongs to the AMPK kinase family and was named after its homolog SIK1, found to be expressed upon high-salt diet-induced stress in rats (14, 15). SIK2 has been implicated into multiple biological roles including melanogenesis, cancer progression, and gluconeogenesis (16–18). SIK2 depletion results in a loss of SCV integrity and bacterial escape into the host cytosol, causing intracellular Salmonella hyperproliferation. Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. Hence, SIK2 may represent a cellular safeguard, which controls the actin cytoskeleton and SCV integrity, thereby serving as a prime regulator of Salmonella proliferation subsequent to cellular internalization.