Nikhil A. Thomas

Expanded Roles for Multicargo and Class 1B Effector Chaperones in Type III Secretion

Bacterial type III secretion systems (T3SS) are complex protein assemblies that mediate the secretion of protein substrates outside the cell. Type III secretion chaperones (T3SC) are always found associated with T3SS, and they serve in multiple roles to ensure that protein substrates are efficiently targeted for secretion. Bacterial pathogens with T3SS express T3SC proteins that bind effectors, a process important for effector protein delivery into eukaryotic cells during infection. In this minireview, we focus on multicargo and class 1B T3SC that associate with effectors within significant pathogens of animals and plants. As a primary role, multicargo and class 1B T3SC form homodimers and specifically bind different effectors within the cytoplasm, maintaining the effectors in a secretion-competent state. This role makes T3SC initial and central contributors to effector-mediated pathogenesis. Recent findings have greatly expanded our understanding of cellular events linked to multicargo T3SC function. New binding interactions with T3SS components have been reported in different systems, thereby implicating multicargo T3SC in critical roles beyond effector binding. Three notable interactions with the YscN, YscV, and YscQ family members are well represented in the literature. Similar T3SC interactions are reported in the putative related flagellar T3SS, suggesting that secretion mechanisms may be more similar than previously thought. The evidence implicates multicargo and class 1B T3SC in effector binding and stabilization, in addition to T3SS recruitment and docking events.

Feed supplementation with red seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, affects performance, egg quality, and gut microbiota of layer hens

The aim of this study was to evaluate the effect of the inclusion of red seaweed supplementation to standard poultry diets on production performance, egg quality, intestinal histology, and cecal short-chain fatty acids in Lohmann Brown Classic laying hens. A total of 160 birds were randomly assigned to 8 treatment groups. Control hens were fed a basal layer diet; positive control hens were fed a diet containing 2% inulin; and 6 treatment groups were fed a diet containing one of the following; 0.5, 1, or 2% Chondrus crispus (CC0.5, CC1, and CC2, respectively) and one of the same 3 levels of Sarcodiotheca gaudichaudii (SG0.5, SG1, and SG2, respectively). Dietary supplementation had no significant effect on the feed intake, BW, egg production, fecal moisture content, and blood serum profile of the birds. The feed conversion ratio per gram of egg was significantly more efficient (P = 0.001) for CC2 and SG2 treatments. Moreover, SG1 supplementation increased egg yolk weight (P = 0.0035) and birds with CC1 supplementation had higher egg weight (P = 0.0006). The SG2 and CC2 groups had greater (P < 0.05) villus height and villus surface area compared with the control birds. Seaweed supplementation increased the abundance of beneficial bacteria [e.g., Bifidobacterium longum (4- to 14-fold), Streptococcus salivarius (4- to 15-fold)] and importantly reduced the prevalence of Clostridium perfringens in the gut of the chicken. Additionally, the concentrations of short-chain fatty acids, including acetic acid, propionic acid, n-butyric acid, and i-butyric acid, were significantly higher (P < 0.05) in CC and SG treatments than in the control. In conclusion, dietary supplementation using red seaweed inclusions can act as a potential prebiotic to improve performance, egg quality, and overall gut health in layer hens.

Role of EscU auto-cleavage in promoting type III effector translocation into host cells by enteropathogenic Escherichia coli

BackgroundType III secretion systems (T3SS) of bacterial pathogens coordinate effector protein injection into eukaryotic cells. The YscU/FlhB group of proteins comprises members associated with T3SS which undergo a specific auto-cleavage event at a conserved NPTH amino acid sequence. The crystal structure of the C-terminal portion of EscU from enteropathogenic Escherichia coli (EPEC) suggests this auto-cleaving protein provides an interface for substrate interactions involved in type III secretion events.ResultsWe demonstrate EscU must be auto-cleaved for bacteria to efficiently deliver type III effectors into infected cells. A non-cleaving EscU(N262A) variant supported very low levels of in vitro effector secretion. These effector proteins were not able to support EPEC infection of cultured HeLa cells. In contrast, EscU(P263A) was demonstrated to be partially auto-cleaved and moderately restored effector translocation and functionality during EPEC infection, revealing an intermediate phenotype. EscU auto-cleavage was not required for inner membrane association of the T3SS ATPase EscN or the ring forming protein EscJ. In contrast, in the absence of EscU auto-cleavage, inner membrane association of the multicargo type III secretion chaperone CesT was altered suggesting that EscU auto-cleavage supports docking of chaperone-effector complexes at the inner membrane. In support of this interpretation, evidence of novel effector protein breakdown products in secretion assays were linked to the non-cleaved status of EscU(N262A).ConclusionsThese data provide new insight into the role of EscU auto-cleavage in EPEC. The experimental data suggests that EscU auto-cleavage results in a suitable binding interface at the inner membrane that accommodates protein complexes during type III secretion events. The results also demonstrate that altered EPEC genetic backgrounds that display intermediate levels of effector secretion and translocation can be isolated and studied. These genetic backgrounds should be valuable in deciphering sequential and temporal events involved in EPEC type III secretion.

The Gut Microbiome of Pediatric Crohn's Disease Patients Differs from Healthy Controls in Genes That Can Influence the Balance Between a Healthy and Dysregulated Immune Response.

Background:Exclusive enteral nutrition (EEN) is a first-line therapy in pediatric Crohns disease (CD) thought to induce remission through changes in the gut microbiome. With microbiome assessment largely focused on microbial taxonomy and diversity, it remains unclear to what extent EEN induces functional changes that thereby contribute to its therapeutic effect. Methods:Fecal samples were collected from 15 pediatric CD patients prior to and after EEN treatment, as well as from 5 healthy controls. Metagenomic data were obtained via next-generation sequencing, and nonhuman reads were mapped to KEGG pathways, where possible. Pathway abundance was compared between CD patients and controls, and between CD patients that sustained remission (SR) and those that did not sustain remission (NSR). Results:Of 132 KEGG pathways identified, 8 pathways differed significantly between baseline CD patients and controls. Examination of these eight pathways showed SR patients had greater similarity to controls than NSR patients in all cases. Pathways fell into one of three groups: 1) no prior connection to IBD, 2) previously reported connection to IBD, and 3) known roles in innate immunity and immunoregulation. Conclusions:The microbiota of CD patients and controls represent alternative ecological states that have broad differences in functional capabilities, including xenobiotic and environmental pollutant degradation, succinate metavolism, and bacterial HtpG, all of which can affect barrier integrity and immune regulation. Moreover, our finding that SR patients were more similar to healthy controls suggests that community microbial function, as inferred from fecal microbiomes, could serve as a valuable diagnostic tool.

Early Changes in Microbial Community Structure Are Associated with Sustained Remission After Nutritional Treatment of Pediatric Crohn's Disease.

Background:Clinical remission achieved by exclusive enteral nutrition (EEN) is associated with marked microbiome changes. In this prospective study of exclusive enteral nutrition, we employ a hierarchical model of microbial community structure to distinguish between pediatric Crohns disease patients who achieved sustained remission (SR) and those who relapsed early (non-SR), after restarting a normal diet. Methods:Fecal samples were obtained from 10 patients (age 10–16) and from 5 healthy controls (age 9–14). The microbiota was assessed via 16S rRNA sequencing. In addition to standard measures of microbial biodiversity, we employed Bayesian methods to characterize the hierarchical community structure. Community structure between patients who sustained remission (wPCDAI <12.5) up to their 24-week follow-up (SR) was compared with patients that had not sustained remission (non-SR). Results:Microbial diversity was lower in Crohns disease patients relative to controls and lowest in patients who did not achieve SR. SR patients differed from non-SR patients in terms of the structure and prevalence of their microbial communities. The SR prevalent community contained a number of strains of Akkermansia muciniphila and Bacteroides and was limited in Proteobacteria, whereas the non-SR prevalent community had a large Proteobacteria component. Their communities were so different that a model trained to discriminate SR and non-SR had 80% classification accuracy, already at baseline sampling. Conclusions:Microbial community structure differs between healthy controls, patients who have an enduring response to exclusive enteral nutrition, and those who relapse early on introduction of normal diet. Our novel Bayesian approach to these differences is able to predict sustained remission after exclusive enteral nutrition.

Properdin Provides Protection from Citrobacter rodentium–Induced Intestinal Inflammation in a C5a/IL-6–Dependent Manner

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium–induced colitis. Following infection, properdin knockout (PKO) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell–derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to PKO mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of PKO mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.

Red Seaweeds Sarcodiotheca gaudichaudii and Chondrus crispus down Regulate Virulence Factors of Salmonella Enteritidis and Induce Immune Responses in Caenorhabditis elegans.

Red seaweeds are a rich source of unique bioactive compounds and secondary metabolites that are known to improve human and animal health. S. Enteritidis is a broad range host pathogen, which contaminates chicken and poultry products that end into the human food chain. Worldwide, Salmonella outbreaks have become an important economic and public health concern. Moreover, the development of resistance in Salmonella serovars toward multiple drugs highlights the need for alternative control strategies. This study evaluated the antimicrobial property of red seaweeds extracts against Salmonella Enteritidis using the Caenorhabditis elegans infection model. Six red seaweed species were tested for their antimicrobial activity against S. Enteritidis and two, Sarcodiotheca gaudichaudii (SG) and Chondrus crispus (CC), were found to exhibit such properties. Spread plate assay revealed that SG and CC (1%, w/v) significantly reduced the growth of S. Enteritidis. Seaweed water extracts (SWE) of SG and CC, at concentrations from 0.4 to 2 mg/ml, significantly reduced the growth of S. Enteritidis (log CFU 4.5–5.3 and log 5.7–6.0, respectively). However, methanolic extracts of CC and SG did not affect the growth of S. Enteritidis. Addition of SWE (0.2 mg/ml, CC and SG) significantly decreased biofilm formation and reduced the motility of S. Enteritidis. Quantitative real-time PCR analyses showed that SWE (CC and SG) suppressed the expression of quorum sensing gene sdiA and of Salmonella Pathogenesis Island-1 (SPI-1) associated genes sipA and invF, indicating that SWE might reduce the invasion of S. Enteritidis in the host by attenuating virulence factors. Furthermore, CC and SG water extracts significantly improved the survival of infected C. elegans by impairing the ability of S. Enteritidis to colonize the digestive tract of the nematode and by enhancing the expression of C. elegans immune responsive genes. As the innate immune response pathways of C. elegans and mammals show a high degree of conservation, these results suggest that these SWE may also impart beneficial effects on animal and human health.

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli.

The locus of enterocyte effacement (LEE) is a 35 kb pathogenicity island involved in attaching and effacing (A/E) Escherichia coli enteric infection. The LEE is organized into five large transcriptional operons (LEE1-LEE5) and a few bi- and monocistronic instances. The LEE5 operon co-transcribes three genes, tir-cesT-eae, although cesT can be transcribed in a separate mRNA from its own proximal promoter. The role of this separate promoter is not understood. In this study we characterized promoter activity for the type III secretion chaperone gene cesT. The cesT promoter, cesTp, has features consistent with σ(70) promoters that contain an extended -10 element. This was experimentally confirmed by mutations that altered cesTp activity. In stark contrast to LEE2-5 transcriptional operons, cesTp did not require the master regulator Ler for transcriptional activity. Moreover, cesTp activity was not dependent on the presence of GrlA or GrlR, two regulators associated with LEE gene expression. A cesTp-lux fusion was used in real-time assays and demonstrated initial cesTp activity that occurred before LEE5 promoter activity, which ensued after 120 min. cesTp was shown to be active during in vitro infection of HT-29 colonic epithelial cells. Inactivation of cesTp reduced CesT protein levels at early growth time points. These data indicate a Ler-, GrlA- and GrlR-independent activity for cesTp. We suggest that A/E pathogenic E. coli have evolved a mechanism to ready the cell for CesT protein expression in support of infection prior to Ler- and GrlA-related activities.

Feed Supplementation with Red Seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, Reduce Salmonella Enteritidis in Laying Hens

Salmonella Enteritidis is vertically transmitted to eggs from laying hens through infected ovaries and oviducts. S. Enteritidis can also penetrate the eggshell from contaminated feces. Reducing S. Enteritidis in laying hens is vital to provide safer eggs and minimize the spread of salmonellosis to humans. Antibiotics have been widely used to control bacterial diseases in broilers and laying hens. However, there is a major concern that the use of antibiotics leads to the development of antibiotic resistance and adverse effects on microbiota of the treated birds. Thus, there is an interest in developing alternatives to antibiotics, such as dietary prebiotics. In the present study, feed supplemented with the red seaweeds: Chondrus crispus (CC) or Sarcodiotheca gaudichaudii (SG), was offered to laying hens late in production to control S. Enteritidis. Diets contained one of the following; 2% or 4% Chondrus crispus (CC2, and CC4, respectively) or Sarcodiotheca gaudichaudii (SG2 and SG4, respectively). Chlortetracycline was used in the positive control diet. During week-4, 48 birds were orally challenged with 2 × 109 CFU/mL of S. Enteritidis. Eggs and fecal samples were collected 1, 3, 5, and 7 days’ post inoculation. Birds were euthanized and organs (ceca, ovary, liver, and spleen) were sampled and analyzed for the presence of S. Enteritidis, 7 days’ post inoculation. Results showed that seaweed reduced the negative effect on body weight and egg production in S. Enteritidis-challenged laying hens. Analysis of fecal samples showed that the antibiotic (CTC) reduced S. Enteritidis in the intestinal tract and fecal samples, 3 days’ post inoculation. Fecal samples from Chlortetracycline and CC4 supplemented birds tested negative for S. Enteritidis on days 5 and 7 post inoculation (lowest detection limit = 10-1). S. Enteritidis colonization in the ceca was also significantly reduced in birds fed CC (4%) and Chlortetracycline. Blood serum profiles revealed that there were no significant differences in serum aspartate aminotransferase (AST) and sodium. However, the level of serum immunoglobulin (IgA) was higher in the CC4 treatment. The relative abundance of Lactobacillus acidophilus was significantly higher in CC4 while, the abundance of the pathogenic bacteria, Clostridium perfringens and Salmonella Enteritidis were reduced compared to control. Results indicate that feed supplemented with 4% CC is effective in providing protection against Salmonella Enteritidis colonization in laying hens.