Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses.

Chikungunya (CHIKV), o'nyong-nyong (ONNV), and Mayaro (MAYV) viruses are transmitted by mosquitoes and known to cause a debilitating arthritogenic syndrome. These alphaviruses have emerged and re-emerged, leading to outbreaks in tropical and subtropical regions of Asia, South America, and Africa. Despite their prevalence, there persists a critical gap in the availability of sensitive and virus-specific point-of-care (POC) diagnostics. Traditional immunoglobulin-based tests such as enzyme-linked immunosorbent assay (ELISAs) often yield cross-reactive results due to the close genetic relationship between these viruses. Molecular diagnostics such as quantitative polymerase chain reaction (qPCR) offer high sensitivity but are limited by the need for specialized laboratory equipment. Recombinase polymerase amplification (RPA), an isothermal amplification method, is a promising alternative to qPCR, providing rapid results with minimal equipment requirements. Here, we report the development and validation of three virus-specific RPA-based POC tests for CHIKV, ONNV, and MAYV. These tests demonstrated both speed and sensitivity, capable of detecting 10 viral copies within 20 minutes of amplification, without exhibiting cross-reactivity. Furthermore, we evaluated the clinical potential of these tests using serum and tissue samples from CHIKV, ONNV, and MAYV-infected mice, as well as CHIKV-infected human patients. We demonstrate that the RPA amplicons derived from the patient samples can be sequenced, enabling cost-effective molecular epidemiological studies. Our findings highlight the significance of these rapid and specific POC diagnostics in improving the early detection and management of these arboviral infections.

Advancing arbovirus diagnosis in Brazil: strengthening diagnostic strategies and public health data collection.

BACKGROUND: The last five decades have seen a surge in viral outbreaks, particularly in tropical and subtropical regions like Brazil, where endemic arboviruses such as Dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) pose significant threats. However, current diagnostic strategies exhibit limitations, leading to gaps in infection screening, arbovirus differential diagnoses, DENV serotyping, and life-long infection tracking. This deficiency impedes critical information availability regarding an individual's current infection and past infection history, disease risk assessment, vaccination needs, and policy formulation. Additionally, the availability of point-of-care diagnostics and knowledge regarding immune profiles at the time of infection are crucial considerations. OBJECTIVES: This review underscores the urgent need to strengthen diagnostic methods for arboviruses in Brazil and emphasizes the importance of data collection to inform public health policies for improved diagnostics, surveillance, and policy formulation. METHODS: We evaluated the diagnostic landscape for arboviral infections in Brazil, focusing on tailored, validated methods. We assessed diagnostic methods available for sensitivity and specificity metrics in the context of Brazil. RESULTS: Our review identifies high-sensitivity, high-specificity diagnostic methods for arboviruses and co-infections. Grifols transcription-mediated amplification assays are recommended for DENV, CHIKV, and ZIKV screening, while IgG/IgM ELISA assays outperform Rapid Diagnostic Tests (RDTs). The Triplex real-time RT-PCR assay is recommended for molecular screening due to its sensitivity and specificity. CONCLUSION: Enhanced diagnostic methods, on-going screening, and tracking are urgently needed in Brazil to capture the complex landscape of arboviral infections in the country. Recommendations include nationwide arbovirus differential diagnosis for DENV, ZIKV, and CHIKV, along with increased DENV serotyping, and lifelong infection tracking to combat enduring viral threats and reduce severe presentations.

The Adaptive Immune Response against Bunyavirales.

The Bunyavirales order includes at least fourteen families with diverse but related viruses, which are transmitted to vertebrate hosts by arthropod or rodent vectors. These viruses are responsible for an increasing number of outbreaks worldwide and represent a threat to public health. Infection in humans can be asymptomatic, or it may present with a range of conditions from a mild, febrile illness to severe hemorrhagic syndromes and/or neurological complications. There is a need to develop safe and effective vaccines, a process requiring better understanding of the adaptive immune responses involved during infection. This review highlights the most recent findings regarding T cell and antibody responses to the five Bunyavirales families with known human pathogens (Peribunyaviridae, Phenuiviridae, Hantaviridae, Nairoviridae, and Arenaviridae). Future studies that define and characterize mechanistic correlates of protection against Bunyavirales infections or disease will help inform the development of effective vaccines.

High SARS-CoV-2 seroprevalence in Lagos, Nigeria with robust antibody and cellular immune responses.

Background: Early evidence suggested that the impact of the COVID-19 pandemic was less severe in Africa compared to other parts of the world. However, more recent studies indicate higher SARS-CoV-2 infection and COVID-19 mortality rates on the continent than previously documented. Research is needed to better understand SARS-CoV-2 infection and immunity in Africa. Methods: In early 2021, we studied the immune responses in healthcare workers (HCWs) at Lagos University Teaching Hospital (n = 134) and Oxford-AstraZeneca COVID-19 vaccine recipients from the general population (n = 116) across five local government areas (LGAs) in Lagos State, Nigeria. Western blots were used to simultaneously detect SARS-CoV-2 spike and nucleocapsid (N) antibodies (n = 250), and stimulation of peripheral blood mononuclear cells with N followed by an IFN-γ ELISA was used to examine T cell responses (n = 114). Results: Antibody data demonstrated high SARS-CoV-2 seroprevalence of 72·4% (97/134) in HCWs and 60·3% (70/116) in the general population. Antibodies directed to only SARS-CoV-2 N, suggesting pre-existing coronavirus immunity, were seen in 9·7% (13/134) of HCWs and 15·5% (18/116) of the general population. T cell responses against SARS-CoV-2 N (n = 114) were robust in detecting exposure to the virus, demonstrating 87·5% sensitivity and 92·9% specificity in a subset of control samples tested. T cell responses against SARS-CoV-2 N were also observed in 83.3% of individuals with N-only antibodies, further suggesting that prior non-SARS-CoV-2 coronavirus infection may provide cellular immunity to SARS-CoV-2. Conclusions: These results have important implications for understanding the paradoxically high SARS-CoV-2 infection with low mortality rate in Africa and supports the need to better understand the implications of SARS-CoV-2 cellular immunity.

Antibody and Cell-Based Therapies against Virus-Induced Cancers in the Context of HIV/AIDS.

Infectious agents, notably viruses, can cause or increase the risk of cancer occurrences. These agents often disrupt normal cellular functions, promote uncontrolled proliferation and growth, and trigger chronic inflammation, leading to cancer. Approximately 20% of all cancer cases in humans are associated with an infectious pathogen. The International Agency for Research on Cancer (IARC) recognizes seven viruses as direct oncogenic agents, including Epstein-Barr Virus (EBV), Kaposi's Sarcoma-associated herpesvirus (KSHV), human T-cell leukemia virus type-1 (HTLV-1), human papilloma virus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1). Most viruses linked to increased cancer risk are typically transmitted through contact with contaminated body fluids and high-risk behaviors. The risk of infection can be reduced through vaccinations and routine testing, as well as recognizing and addressing risky behaviors and staying informed about public health concerns. Numerous strategies are currently in pre-clinical phases or undergoing clinical trials for targeting cancers driven by viral infections. Herein, we provide an overview of risk factors associated with increased cancer incidence in people living with HIV (PLWH) as well as other chronic viral infections, and contributing factors such as aging, toxicity from ART, coinfections, and comorbidities. Furthermore, we highlight both antibody- and cell-based strategies directed against virus-induced cancers while also emphasizing approaches aimed at discovering cures or achieving complete remission for affected individuals.

Monoclonal antibody pairs against SARS-CoV-2 for rapid antigen test development.

BACKGROUND: The focus on laboratory-based diagnosis of coronavirus disease 2019 (COVID-19) warrants alternative public health tools such as rapid antigen tests. While there are a number of commercially available antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), all cross-react with the genetically similar SARS-CoV-1 or require an instrument for results interpretation. METHODOLOGY/PRINCIPAL FINDINGS: We developed and validated rapid antigen tests that use pairs of murine-derived monoclonal antibodies (mAbs), along with gold nanoparticles, to detect SARS-CoV-2 with or without cross-reaction to SARS-CoV-1 and other coronaviruses. In this development, we demonstrate a robust antibody screening methodology for the selection of mAb pairs that can recognize SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. Linear epitope mapping of the mAbs helped elucidate SARS-CoV-2 S and N interactions in lateral flow chromatography. A candidate rapid antigen test for SARS-CoV-2 N was validated using nasal swab specimens that were confirmed positive or negative by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Test results were image-captured using a mobile phone and normalized signal pixel intensities were calculated; signal intensities were inversely correlated to RT-PCR cycle threshold (Ct) value. CONCLUSION/SIGNIFICANCE: Overall, our results suggest that the rapid antigen test is optimized to detect SARS-CoV-2 N during the acute phase of COVID-19. The rapid antigen tests developed in this study are alternative tools for wide scale public health surveillance of COVID-19.

Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2.

Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.

Development and Validation of a Rapid Screening Test for HTLV-I IgG Antibodies.

Initial diagnosis of human T cell lymphotropic virus (HTLV) infections is mainly based by detecting antibodies in plasma or serum using laboratory-based methods. The aim of this study was to develop and evaluate a rapid screening test for HTLV-I antibodies. Our rapid screening test uses HTLV-I p24 antigen conjugated to gold nanoparticles and an anti-human IgG antibody immobilized to a nitrocellulose strip to detect human HTLV-I p24-specific IgG antibodies via immunochromatography. Performance of the rapid screening test for HTLV-I was conducted on a total of 118 serum specimens collected in Salvador, Bahia, the epicenter for HTLV-1 infection in Brazil. Using a Western blot test as the comparator, 55 serum specimens were HTLV-I positive, 5 were HTLV-I and HTLV-II positive, and 58 were negative. The sensitivity of the rapid screening test for HTLV-1 was 96.7% and the specificity was 100%. The rapid screening test did not show cross-reaction with serum specimens from individuals with potentially interfering infections including those caused by HTLV-II, HIV-I, HIV-II, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, Epstein-Barr virus, SARS-CoV-2, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Toxoplasma gondii, and Plasmodium falciparum. The rapid screening test also did not show cross-reaction with potentially interfering substances. Strategies for HTLV diagnosis in non- and high-endemic areas can be improved with low-cost, rapid screening tests.

Pooling of Samples for SARS-CoV-2 Detection Using a Rapid Antigen Test.

While molecular assays, such as reverse-transcription polymerase chain reaction (RT-PCR), have been widely used throughout the coronavirus disease 2019 (COVID-19) pandemic, the technique is costly and resource intensive. As a means to reduce costs and increase diagnostic efficiency, pooled testing using RT-PCR has been implemented. However, pooling samples for antigen testing has not been evaluated. Here, we propose a proof-of-concept pooling strategy for antigen testing that would significantly expand SARS-CoV-2 surveillance, especially for low-to-middle income countries, schools, and workplaces. Our laboratory-based testing demonstrates that combining of up to 20 nasal swab specimens per pool can expand surveillance with antigen tests, even if a pool contains only one positive sample.

Development and Validation of a Rapid Lateral Flow E1/E2-Antigen Test and ELISA in Patients Infected with Emerging Asian Strain of Chikungunya Virus in the Americas.

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.

Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort.

BACKGROUND: Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1-4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available. METHODOLOGY/PRINCIPLE FINDINGS: We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07-100%. SIGNIFICANCE: Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions.

Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis.

Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a challenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001-2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-antithrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lymphocytes (%) and thrombin time were increased. In dengue patients, the absolute lymphocytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamination events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies.

T Cell Responses to Nonstructural Protein 3 Distinguish Infections by Dengue and Zika Viruses.

The 2015-2016 Zika virus (ZIKV) epidemic in the Americas and the Caribbean demonstrated that clinical assays to detect, distinguish, and characterize immune responses to flaviviral infections are needed. ZIKV and dengue virus (DENV) are mosquito-transmitted flaviviruses sharing overlapping geographic distributions and have significant sequence similarities that can increase the potential for antibody and T cell cross-reaction. Using nonstructural protein 1-based enzyme-linked immunosorbent assays (ELISAs), we determined the serostatus of individuals living in a region of DENV and ZIKV endemicity in Brazil, identifying individuals with primary DENV (pDENV) and primary ZIKV (pZIKV), ZIKV with primary DENV (ZIKVwpDENV), and secondary DENV (sDENV) infections; the presence of pDENV and pZIKV was further confirmed by neutralization tests. Development of an enzyme-linked immunosorbent spot (ELISPOT) assay for DENV and ZIKV structural and nonstructural (NS) protein antigens enabled us to distinguish infections by these viruses based on T cell responses and to characterize those responses. We found that gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) T cell responses to NS3 differentiated DENV and ZIKV infections with 94% sensitivity and 92% specificity. In general, we also showed that pDENV and sDENV cases and pZIKV and ZIKVwpDENV cases elicit similar T cell response patterns and that HIV-infected individuals show T cell responses that are lower than those shown by HIV-negative individuals. These results have important implications for DENV and ZIKV diagnostic and vaccine development and provide critical insights into the T cell response in individuals with multiple flaviviral infections.IMPORTANCE The potential for antibody and T cell cross-reactions to DENV and ZIKV, flaviviruses that cocirculate and can sequentially infect individuals, has complicated diagnostic and vaccine development. Our serological data show that antibodies to nonstructural protein 1 can distinguish sequential human infections by DENV and ZIKV. The development of a simple and inexpensive assay also enables the differentiation of DENV and ZIKV infections based on characterization of T cell responses. Our T cell data reveal strong response patterns that are similar in nature to those seen with individuals with one or multiple DENV infections and with individuals with only primary ZIKV infection and ZIKV-infected individuals with previous DENV exposure. The characterization of T cell responses in a serologically validated group of individuals is of relevance to the development of vaccines and immunotherapeutics against these global threats.

A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals.

BACKGROUND: Ebola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. METHODOLOGY/PRINCIPLE FINDINGS: Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. CONCLUSION/SIGNIFICANCE: Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

Sustained Specific and Cross-Reactive T Cell Responses to Zika and Dengue Virus NS3 in West Africa.

Recent studies on the role of T cells in Zika virus (ZIKV) infection have shown that T cell responses to Asian ZIKV infection are important for protection, and that previous dengue virus (DENV) exposure amplifies the protective T cell response to Asian ZIKV. Human T cell responses to African ZIKV infection, however, remain unexplored. Here, we utilized the modified anthrax toxin delivery system to develop a flavivirus enzyme-linked immunosorbent spot (ELISPOT) assay. Using human ZIKV and DENV samples from Senegal, West Africa, our results demonstrate specific and cross-reactive T cell responses to nonstructural protein 3 (NS3). Specifically, we found that T cell responses to NS3 protease are ZIKV and DENV specific, but responses to NS3 helicase are cross-reactive. Sequential sample analyses revealed immune responses sustained many years after infection. These results have important implications for African ZIKV/DENV vaccine development, as well as for potential flavivirus diagnostics based on T cell responses.IMPORTANCE The recent Zika virus (ZIKV) epidemic in Latin America and the associated congenital microcephaly and Guillain-Barré syndrome have raised questions as to why we have not recognized these distinct clinical diseases in Africa. The human immunologic response to ZIKV and related flaviviruses in Africa represents a research gap that may shed light on the mechanisms contributing to protection. The goal of our study was to develop an inexpensive assay to detect and characterize the T cell response to African ZIKV and DENV. Our data show long-term specific and cross-reactive human immune responses against African ZIKV and DENV, suggesting the usefulness of a diagnostic based on the T cell response. Additionally, we show that prior flavivirus exposure influences the magnitude of the T cell response. The identification of immune responses to African ZIKV and DENV is of relevance to vaccine development.

Continued Transmission of Zika Virus in Humans in West Africa, 1992-2016.

First identified in 1947 in Uganda, Zika virus (ZIKV) has remained largely unstudied until the recent outbreak in Latin America. This study aimed to measure the prevalence of ZIKV in febrile patients in Senegal and Nigeria in samples collected from 1992 to 2016. The seroprevalence of ZIKV was 6.2% based on ZIKV immunoglobulin M and negative for dengue reactivity. ZIKV envelope was amplified from 4 samples. Phylogenetic analysis showed that the ZIKVs belonged to the African lineage, grouping with either the Nigerian or MR766 sublineages. This study provides evidence that ZIKV has been silently circulating in West Africa for 2 decades.

Intermediate filaments enable pathogen docking to trigger type 3 effector translocation.

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

Shigella Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation.

The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.