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Krüppel-like factor 2 (KLF2) is a potent regulator of lymphocyte differentiation, activation and migration. However, its functional role in adaptive and humoral immunity remains elusive. Therefore, by using mice with a B cell-specific deletion of KLF2, we investigated plasma cell differentiation and antibody responses. We revealed that the deletion of KLF2 resulted in perturbed IgA plasma cell compartmentalization, characterized by the absence of IgA plasma cells in the bone marrow, their reductions in the spleen, the blood and the lamina propria of the colon and the small intestine, concomitant with their accumulation and retention in mesenteric lymph nodes and Peyer's patches. Most intriguingly, secretory IgA in the intestinal lumen was almost absent, dimeric serum IgA was drastically reduced and antigen-specific IgA responses to soluble Salmonellaflagellin were blunted in KLF2-deficient mice. Perturbance of IgA plasma cell localization was caused by deregulation of CCR9, Integrin chains αM, α4, β7, and sphingosine-1-phosphate receptors. Hence, KLF2 not only orchestrates the localization of IgA plasma cells by fine-tuning chemokine receptors and adhesion molecules but also controls IgA responses to Salmonellaflagellin.

Krüppel-like factor 2 (KLF2) is a potent regulator of lymphocyte differentiation, activation and migration. However, its functional role in adaptive and humoral immunity remains elusive. Therefore, by using mice with a B cell-specific deletion of KLF2, we investigated plasma cell differentiation and antibody responses. We revealed that the deletion of KLF2 resulted in perturbed IgA plasma cell compartmentalization, characterized by the absence of IgA plasma cells in the bone marrow, their reductions in the spleen, the blood and the lamina propria of the colon and the small intestine, concomitant with their accumulation and retention in mesenteric lymph nodes and Peyer’s patches. Most intriguingly, secretory IgA in the intestinal lumen was almost absent, dimeric serum IgA was drastically reduced and antigen-specific IgA responses to soluble Salmonellaflagellin were blunted in KLF2-deficient mice. Perturbance of IgA plasma cell localization was caused by deregulation of CCR9, Integrin chains αM, α4, β7, and sphingosine-1-phosphate receptors. Hence, KLF2 not only orchestrates the localization of IgA plasma cells by fine-tuning chemokine receptors and adhesion molecules but also controls IgA responses to Salmonellaflagellin.

•PRN473 blocks CLEC-2–mediated platelet function in vitro and ex vivo and reduces thrombosis in 2 mouse models of venous thrombosis.•The data and lack of bleeding in PRN1008/rilzabrutinib clinical trials suggest a future use in immune-mediated thrombosis.

ObjectiveTo determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers.DesignA cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020.SettingUniversity Hospitals Birmingham NHS Foundation Trust (UHBFT), UK.Participants545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded.InterventionParticipants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked.Main outcome measureProportion of participants demonstrating infection and positive SARS-CoV-2 serology.ResultsThe point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02).Conclusions and relevanceWe identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by SalmonellaTyphimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonellain vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3′ untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

Although specific antibody induced by pathogens or vaccines is a key component of protection against infectious threats, some viruses, such as dengue, induce antibody that enhances the development of infection. In contrast, antibody-dependent enhancement of bacterial infection is largely unrecognized. Here, we demonstrate that in a significant portion of patients with bronchiectasis and Pseudomonas aeruginosa lung infection, antibody can protect the bacterium from complement-mediated killing. Strains that resist antibody-induced, complement-mediated killing produce lipopolysaccharide containing O-antigen. The inhibition of antibody-mediated killing is caused by excess production of O-antigen–specific IgG2 antibodies. Depletion of IgG2 to O-antigen restores the ability of sera to kill strains with long-chain O-antigen. Patients with impaired serum-mediated killing of P. aeruginosa by IgG2 have poorer respiratory function than infected patients who do not produce inhibitory antibody. We suggest that excessive binding of IgG2 to O-antigen shields the bacterium from other antibodies that can induce complement-mediated killing of bacteria. As there is significant sharing of O-antigen structure between different Gram-negative bacteria, this IgG2-mediated impairment of killing may operate in other Gram-negative infections. These findings have marked implications for our understanding of protection generated by natural infection and for the design of vaccines, which should avoid inducing such blocking antibodies.

Type I secretion systems (T1SS) are versatile molecular machines for protein transport across the Gram-negative cell envelope. The archetypal Type I system mediates secretion of the Escherichia colihemolysin, HlyA. This system has remained the pre-eminent model of T1SS research since its discovery. The classic description of a T1SS is composed of three proteins: an inner membrane ABC transporter, a periplasmic adaptor protein and an outer membrane factor. According to this model, these components assemble to form a continuous channel across the cell envelope, an unfolded substrate molecule is then transported in a one-step mechanism, directly from the cytosol to the extracellular milieu. However, this model does not encapsulate the diversity of T1SS that have been characterized to date. In this review, we provide an updated definition of a T1SS, and propose the subdivision of this system into five subgroups. These subgroups are categorized as T1SSa for RTX proteins, T1SSb for non-RTX Ca2+-binding proteins, T1SSc for non-RTX proteins, T1SSd for class II microcins, and T1SSe for lipoprotein secretion. Although often overlooked in the literature, these alternative mechanisms of Type I protein secretion offer many avenues for biotechnological discovery and application.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell–dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell–dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

ABSTRACTSalmonella entericais a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonellaare of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella entericaserovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonellacell surface in vitroand in vivoduring infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against SALMONELLA:

Mutations in σE-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during in vivoinfection. YraP is conserved across a number of Gram-negative pathogens, including Neisseria meningitidis, where the homolog is a component of the Bexsero meningococcal group B vaccine.

ABSTRACTAntibodies acquired after vaccination or natural infection with Gram-negative bacteria, such as invasive Salmonella entericaserovar Typhimurium, can protect against disease. Immunization with naturally shed outer membrane vesicles from Gram-negative bacteria is being studied for its potential to protect against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification, and the resulting particles, generalized modules for membrane antigens (GMMA), not only offer potential as vaccines but also can facilitate the study of B-cell responses to bacterial antigens. Here we show that the response to immunization with GMMA from S. Typhimurium (STmGMMA) provides B-cell-dependent protection and induces antibodies to two immunodominant antigens, lipopolysaccharide (LPS) and porins. Antibodies to LPS O antigen (O-Ag) markedly enhance protection in the spleen, but this effect is less marked in the liver. Strikingly, IgG responses to LPS and porins develop with distinct kinetics. In the first week after immunization, there is a dramatic T-cell-independent B1b-cell-associated induction of all IgG isotypes, except IgG1, to porins but not to LPS. In contrast, production of IgG1 to either antigen was delayed and T cell dependent. Nevertheless, after 1 month, cells in the bone marrow secreting IgG against porins or LPS were present at a similar frequency. Unexpectedly, immunization with O-Ag-deficient STmGMMA did not substantially enhance the anti-porin response. Therefore, IgG switching to all antigens does not develop synchronously within the same complex and so the rate of IgG switching to a single component does not necessarily reflect its frequency within the antigenic complex.IMPORTANCEVaccines save millions of lives, yet for some infections there are none. This includes some types of Salmonellainfections, killing hundreds of thousands of people annually. We show how a new type of vaccine, called GMMA, that is made from blebs shed from the Salmonellacell wall, works to protect against infection in mice by inducing host proteins (antibodies) specifically recognizing bacterial components (antigens). The rate of development of IgG antibody to antigens within GMMA occurred with different kinetics. However, the antibody response to GMMA persists and is likely to provide prolonged protection for those who need it. These results help show how antibody responses to bacterial antigens develop and how vaccines like GMMA can work and help prevent infection.

ABSTRACTTransposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coliK-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. colimodel laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.

ABSTRACTThe use of recombinant attenuated Salmonellavaccine (RASV) strains is a promising strategy for presenting heterologous antigens to the mammalian immune system to induce both cellular and humoral immune responses. However, studies on RASV development differ on where heterologous antigens are expressed and localized within the bacterium, and it is unclear how antigen localization modulates the immune response. Previously, we exploited the plasmid-encoded toxin (Pet) autotransporter system for accumulation of heterologous antigens in cell culture supernatant. In the present study, this Pet system was used to express early secretory antigen 6 (ESAT-6), an immunodominant and diagnostic antigen from Mycobacterium tuberculosis, in Salmonella entericaserovar Typhimurium strain SL3261. Three strains were generated, whereby ESAT-6 was expressed as a cytoplasmic (SL3261/cyto), surface-bound (SL3261/surf), or secreted (SL3261/sec) antigen. Using these RASVs, the relationship between antigen localization and immunogenicity in infected C57BL/6 mice was systematically examined. Using purified antigen and specific tetramers, we showed that mice infected with the SL3261/surf or SL3261/sec strain generated large numbers of Th1 CD4+ESAT-6+splenic T cells compared to those of mice infected with SL3261/cyto. While all mice showed ESAT-6-specific antibody responses when infected with SL3261/surf or SL3261/sec, peak total serum IgG antibody titers were reached more rapidly in mice that received SL3261/sec. Thus, how antigen is localized after production within bacteria has a more marked effect on the antibody response than on the CD4+T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs.