Shigella Vaccines: The Continuing Unmet Challenge.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

Development of a visual Adhesion/Invasion Inhibition Assay to assess the functionality of Shigella-specific antibodies.

Introduction: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella's unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level. Objectives and methods: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix. Results: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner. Discussion: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.

Recent advances in modelling Shigella infection.

Shigella is an important human-adapted pathogen which contributes to a large global burden of diarrhoeal disease. Together with the increasing threat of antimicrobial resistance and lack of an effective vaccine, there is great urgency to identify novel therapeutics and preventatives to combat Shigella infection. In this review, we discuss the development of innovative technologies and animal models to study mechanisms underlying Shigella infection of humans. We examine recent literature introducing (i) the organ-on-chip model, and its substantial contribution towards understanding the biomechanics of Shigella infection, (ii) the zebrafish infection model, which has delivered transformative insights into the epidemiological success of clinical isolates and the innate immune response to Shigella, (iii) a pioneering oral mouse model of shigellosis, which has helped to discover new inflammasome biology and protective mechanisms against shigellosis, and (iv) the controlled human infection model, which has been effective in translating basic research into human health impact and assessing suitability of novel vaccine candidates. We consider the recent contributions of each model and discuss where the future of modelling Shigella infection lies.

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs.

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations.

Morphological, biological, and genomic characterization of a newly isolated lytic phage Sfk20 infecting Shigella flexneri, Shigella sonnei, and Shigella dysenteriae1.

Shigellosis, caused by Shigella bacterial spp., is one of the leading causes of diarrheal morbidity and mortality. An increasing prevalence of multidrug-resistant Shigella species has revived the importance of bacteriophages as an alternative therapy to antibiotics. In this study, a novel bacteriophage, Sfk20, has been isolated from water bodies of a diarrheal outbreak area in Kolkata (India) with lytic activity against many Shigella spp. Phage Sfk20 showed a latent period of 20 min and a large burst size of 123 pfu per infected cell in a one-step growth analysis. Phage-host interaction and lytic activity confirmed by phage attachment, intracellular phage development, and bacterial cell burst using ultrathin sectioning and TEM analysis. The genomic analysis revealed that the double-stranded DNA genome of Sfk20 contains 164,878 bp with 35.62% G + C content and 241 ORFs. Results suggested phage Sfk20 to include as a member of the T4 myoviridae bacteriophage group. Phage Sfk20 has shown anti-biofilm potential against Shigella species. The results of this study imply that Sfk20 has good possibilities to be used as a biocontrol agent.

Pathogens Associated With Linear Growth Faltering in Children With Diarrhea and Impact of Antibiotic Treatment: The Global Enteric Multicenter Study.

BACKGROUND: The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment. METHODS: The Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models. RESULTS: Among 8077 MSD cases analyzed, the proportion with stunting (HAZ below -1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0-11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12-23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02). CONCLUSIONS: Linear growth faltering among children aged 0-23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

Convulsions in children hospitalized for acute gastroenteritis.

The study aim was to examine possible correlates of convulsions in children hospitalized for acute gastroenteritis (AGE). Data collected in a prospective study of AGE hospitalizations in children aged 0-59 months in 3 hospitals in Israel during 2008-2015 were analyzed. Stool samples were tested for rotavirus using immunochromatography and stool culture was performed for the detection of Salmonella, Shigella and Campylobacter We compared clinical and demographic characteristics of children hospitalized for AGE who had convulsions (n = 68, cases) with children hospitalized for AGE without convulsions (n = 3505, controls). Age differed between children with and without convulsions (p = 0.005); the former were mostly toddlers aged 12-23 months (51%) compared to 30% of the control group. A higher percentage of cases tested positive for Shigella (11% vs. 4%, p = 0.002), the opposite was found for rotavirus (2% vs. 30% p < 0.001). A multivariable model showed that body temperature (OR 2.91 [95% CI 1.78-4.76], p < 0.001) and high blood glucose level (> 120 mg/dL) (OR 5.71 [95% CI 1.27-25.58] p = 0.023) were positively related to convulsions in children with AGE, while severe AGE (Vesikari score ≥ 11) was inversely related with convulsions (OR 0.09 [95% CI 0.03-0.24], p < 0.001). Conclusion: Elevated body temperature is associated with convulsions in children with AGE, but not severity of AGE, while hyperglycemia might reflect a neuroendocrine stress reaction to convulsions, AGE or both.

Chemical Profile, Antioxidant Properties and Antimicrobial Activities of Malaysian Heterotrigona itama Bee Bread.

The aim of the study was to determine the chemical profile, antioxidant properties and antimicrobial activities of Heterotrigona itama bee bread from Malaysia. The pH, presence of phytochemicals, antioxidant properties, total phenolic content (TPC) and total flavonoid content (TFC), as well as antimicrobial activities, were assessed. Results revealed a decrease in the pH of bee bread water extract (BBW) relative to bee bread ethanolic extract (BBE) and bee bread hot water extract (BBH). Further, alkaloids, flavonoids, phenols, tannins, saponins, terpenoids, resins, glycosides and xanthoproteins were detected in BBW, BBH and BBE. Also, significant decreases in TPC, TFC, DPPH activity and FRAP were detected in BBW relative to BBH and BBE. We detected phenolic acids such as gallic acid, caffeic acid, trans-ferulic acid, trans 3-hydroxycinnamic acid and 2-hydroxycinnamic acid, and flavonoids such as quercetin, kaempferol, apigenin and mangiferin in BBE using high-performance liquid chromatography analysis. The strongest antimicrobial activity was observed in Klebsilla pneumonia (MIC50 1.914 µg/mL), followed by E. coli (MIC50 1.923 µg/mL), Shigella (MIC50 1.813 µg/mL) and Salmonella typhi (MIC50 1.617 µg/mL). Bee bread samples possess antioxidant and antimicrobial properties. Bee bread contains phenolic acids and flavonoids, and could be beneficial in the management and treatment of metabolic diseases.

New methods to decrypt emerging macropinosome functions during the host-pathogen crosstalk.

Large volumes of liquid and other materials from the extracellular environment are internalised by eukaryotic cells via an endocytic process called macropinocytosis. It is now recognised that this fundamental and evolutionarily conserved pathway is hijacked by numerous intracellular pathogens as an entry portal to the host cell interior. Yet, an increasing number of additional cellular functions of macropinosomes in pathologic processes have been reported beyond this role for fluid internalisation. It emerges that the identity of macropinosomes can vary hugely and change rapidly during their lifetime. A deeper understanding of this important multi-faceted compartment is based on novel methods for their investigation. These methods are either imaging-based for the tracking of macropinosome dynamics, or they provide the means to extract macropinosomes at high purity for comprehensive proteomic analyses. Here, we portray these new approaches for the investigation of macropinosomes. We document how these method developments have provided insights for a new understanding of the intracellular lifestyle of the bacterial pathogens Shigella and Salmonella. We suggest that a systematic complete characterisation of macropinosome subversion with these approaches during other infection processes and pathologies will be highly beneficial for our understanding of the underlying cellular and molecular processes.

Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens.

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Distribution of genes encoding virulence factors of Shigella strains isolated from children with diarrhea in southwest Iran.

This study aimed to investigate the distribution of virulence factor genes in Shigella strains isolated from children with diarrhea in the southwest, Iran. In this cross-sectional study, 1530 diarrheal stool specimens were collected from children aged under 15 years. The Shigella strains were identified by biochemical methods and polymerase chain reaction (PCR). Subsequently, all Shigella isolates were evaluated by PCR for the presence of nine virulence genes ipaH (responsible for dissemination from cell to cell), ial (responsible for epithelial cell penetration), sat (displays cytopathic activity in several intestinal cell lines), sigA (toxic to epithelial cells), pic (associated with colonization), pet (cytotoxic for epithelial cells), sepA (contribute to intestinal inflammation and colonization), virF and invE (regulatory proteins). A total of 91 isolates including 47 S. flexneri, 36 S. sonnei, and 8 S. boydii were identified. All isolates were positive for the ipaH gene. The other genes include ial, virF, invE, sigA, sat, sepA, pic and pet found in 84.6%, 72.5%, 68.1%, 62.6%, 51.6%, 39.5%, 37.3% and 28.5% of the isolates, respectively. The results showed a high distribution of virulence genes among Shigella strains in our region. It seems that for different Shigella spp. different virulence factors contribute to pathogenesis. The current study provided insights into some baseline information about the distribution of some virulence genes of Shigella isolates in Southwest Iran.

Assessment of knowledge, practice, and status of food handlers toward Salmonella, Shigella, and intestinal parasites: A cross-sectional study in Tigrai prison centers, Ethiopia.

BACKGROUND: Unsafe food becomes a global public health and economic threat to humans. The health status, personal hygiene, knowledge, and practice of food handlers have crucial impact on food contamination. Hence, this study is aimed at assessing the knowledge, practice, and prevalence of Salmonella, Shigella, and intestinal parasites among food handlers in Eastern Tigrai prison centers, Northern Ethiopia. METHODS: An institutional-based cross-sectional study was carried out from April to September 2019 among food handlers in Eastern Tigrai prison centers, Northern Ethiopia. A structured questionnaire was used to collect the demographic characteristics, the knowledge, and the practice of the study participants. Direct wet mount and formol-ether concentration techniques were applied to identify intestinal parasites. Culture and biochemical tests were used to isolate the Salmonella and the Shigella species. Additionally, antimicrobial susceptibility tests to selected antibiotics were performed using Kirby-Baur disk diffusion method. We used SPSS version 23 software for statistical analysis. RESULTS: Thirty-seven (62.7%, 37/59) of the participants had harbored one or more intestinal parasites. The protozoan Entamoeba histolytica/dispar was detected among 23.7% (14/59) of the study participants who provided stool specimen. Besides, 6.8% (4/59) of the samples were positive for either Salmonella or Shigella species. The Salmonella isolates (n = 2) were sensitive to Gentamicin, Ciprofloxacin, Ceftriaxone, and Clarithromycin but resistant to Amoxicillin, Ampicillin, and Amoxicillin/clavulanic acid. Similarly, the two Shigella isolates were susceptible to Gentamicin, Ciprofloxacin, and Ceftriaxone but showed resistance to Amoxicillin, Tetracycline, and Chloramphenicol. Further, 60.6% (40/66) of the participants had good level of knowledge, and 51.5% (34/66) had good level of practice on foodborne diseases and on food safety. CONCLUSIONS: We conclude that foodborne pathogens are significant health problems in the study areas. Regular health education and training programs among the food handlers are demanded to tackle foodborne diseases at the prison centers.

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

Comparison of Antimicrobial Resistance Detected in Environmental and Clinical Isolates from Historical Data for the US.

Antimicrobial resistance (AMR) has become an urgent public health issue, as pathogens are becoming increasingly resistant to commonly used antimicrobials. While AMR isolate data are available in the NCBI Pathogen Detection Isolates Browser (NPDIB) database, few researches have been performed to compare antimicrobial resistance detected in environmental and clinical isolates. To address this, this work conducted the first multivariate statistical analysis of antimicrobial-resistance pathogens detected in NPDIB clinical and environmental isolates for the US from 2013 to 2018. The highly occurring AMR genes and pathogens were identified for both clinical and environmental settings, and the historical profiles of those genes and pathogens were then compared for the two settings. It was found that Salmonella enterica and E. coli and Shigella were the highly occurring AMR pathogens for both settings. Additionally, the genes fosA, oqxB, ble, floR, fosA7, mcr-9.1, aadA1, aadA2, ant(2")-Ia, aph(3")-Ib, aph(3')-Ia, aph(6)-Id, blaTEM-1, qacEdelta1, sul1, sul2, tet(A), and tet(B) were mostly detected for both clinical and environmental settings. Ampicillin, ceftriaxone, gentamicin, tetracycline, and cefoxitin were the antimicrobials which got the most resistance in both settings. The historical profiles of these genes, pathogens, and antimicrobials indicated that higher occurrence frequencies generally took place earlier in the environmental setting than in the clinical setting.

Molecular characterization of Shigella species isolated from diarrheal patients in Tehran, Iran: phylogenetic typing and its association with virulence gene profiles and a novel description of Shigella invasion associated locus.

The present study aims to employ a multiplex PCR-based method for phylogenetic typing of Shigella and determine the frequency of several virulence genes among Shigella phylogenetic clades and species. Species identification, phylogenetic typing of 44 previously diagnosed Shigella isolates, and frequency of virulence genes and loci, virA, virB, virF, ipaBCD, ial, sen, and set1A were investigated through performing several PCR assays. Distribution of virulence genes among Shigella phylogenetic clades and species was determined by the statistical analysis. The identities of 40 isolates out of 44 were confirmed as Shigella, and these isolates were classified in four phylogenetic clades, S1 (7.5%), S2 (52.5%), S3 (20%), and S5 (20%) and 4 species, S. sonnei (52.5%), S. flexneri (22.5%), S. dysenteriae (20%), and S. boydii (5%). The prevalence of virA, virB, virF, ipaBCD, ial, sen, and set1A was determined as 67.5%, 72.5%, 72.5%, 65%, 75%, 40%, and 5%, respectively. The presence of sen, uidA, or set1A was found to be statistically correlated with either of Shigella phylogenetic clades or species. A significant statistically association was also determined between set1A and Shigella phylogenetic clades. Furthermore, the nucleotide sequence of invasion-associated locus (ial) was determined and mapped on Shigella genome through in silico analysis. The current study shows the distribution of Shigella isolates and its key virulence genes within the five recently described phylogenetic clades for the first time in the Asia. This is also the first description of ial nucleotide sequence and its exact location on Shigella genome after its initial identification.

Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy.

Shiga toxin (STx) produced by Shigella and closely related Shiga toxin 1 and 2 (STx1 and STx2) synthesized by Shiga toxin-producing Escherichia coli (STEC) are bacterial AB5 toxins. All three toxins target kidney cells and may cause life-threatening renal disease. While Shigella infections can be treated with antibiotics, resistance is increasing. Moreover, antibiotic therapy is contraindicated for STEC, and there are no definitive treatments for STEC-induced disease. To exert cellular toxicity, STx, STx1, and STx2 must undergo retrograde trafficking to reach their cytosolic target, ribosomes. Direct transport from early endosomes to the Golgi apparatus is an essential step that allows the toxins to bypass degradative late endosomes and lysosomes. The essentiality of this transport step also makes it an ideal target for the development of small-molecule inhibitors of toxin trafficking as potential therapeutics. Here, we review the recent advances in understanding the molecular mechanisms of the early endosome-to-Golgi transport of STx, STx1, and STx2, as well as the development of small-molecule inhibitors of toxin trafficking that act at the endosome/Golgi interface.

Comparing the performance of conventional PCR, RTQ-PCR, and droplet digital PCR assays in detection of Shigella.

The incidence of foodborne infections caused by Shigella spp. is still very high in every year, which poses a great potential threat to public health. Conventional quantification methods based on culture techniques, biochemical, and serological identification are time-consuming and labor-intensive. To develop a more rapid and efficient detection method of Shigella spp., we compared the sensitivity and specificity of three different polymerase chain reaction (PCR) methods, including conventional PCR, quantitative real-time PCR (RTQ-PCR), and droplet digital PCR (ddPCR). Our results indicated that ddPCR method exhibited higher sensitivity, and the limit of detection was 10-5 ng/µl for genomic DNA templates, 10-1 cfu/ml for Shigella bacteria culture. In addition, we found that ddPCR was a time-saving method, which required a shorter pre-culturing time. Collectively, ddPCR assay was a reliable method for rapid and effective detection of Shigella spp.

Pathogen-Specific Impacts of the 2011-2012 La Niña-Associated Floods on Enteric Infections in the MAL-ED Peru Cohort: A Comparative Interrupted Time Series Analysis.

Extreme floods pose multiple direct and indirect health risks. These risks include contamination of water, food, and the environment, often causing outbreaks of diarrheal disease. Evidence regarding the effects of flooding on individual diarrhea-causing pathogens is limited, but is urgently needed in order to plan and implement interventions and prioritize resources before climate-related disasters strike. This study applied a causal inference approach to data from a multisite study that deployed broadly inclusive diagnostics for numerous high-burden common enteropathogens. Relative risks (RRs) of infection with each pathogen during a flooding disaster that occurred at one of the sites-Loreto, Peru-were calculated from generalized linear models using a comparative interrupted time series framework with the other sites as a comparison group and adjusting for background seasonality. During the early period of the flood, increased risk of heat-stable enterotoxigenic E. coli (ST-ETEC) was identified (RR = 1.73 [1.10, 2.71]) along with a decreased risk of enteric adenovirus (RR = 0.36 [0.23, 0.58]). During the later period of the flood, sharp increases in the risk of rotavirus (RR = 5.30 [2.70, 10.40]) and sapovirus (RR = 2.47 [1.79, 3.41]) were observed, in addition to increases in transmission of Shigella spp. (RR = 2.86 [1.81, 4.52]) and Campylobacter spp. (RR = 1.41 (1.01, 1.07). Genotype-specific exploratory analysis reveals that the rise in rotavirus transmission during the flood was likely due to the introduction of a locally atypical, non-vaccine (G2P[4]) strain of the virus. Policy-makers should target interventions towards these pathogens-including vaccines as they become available-in settings where vulnerability to flooding is high as part of disaster preparedness strategies, while investments in radical, transformative, community-wide, and locally-tailored water and sanitation interventions are also needed.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence.

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.