The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis , unlike the case with E. coli , cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response. Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterial pathogen that invades professional phagocytes and other host cells to cause scrub typhus, a potentially deadly human disease that significantly impacts global health. How O. tsutsugamushi modulates immune signaling is poorly understood. In this study, we demonstrate that O. tsutsugamushi inhibits the accumulation of the NF-κB subunit, p65, in the nucleus and NF-κB mediated transcription, both of which are critical for the antimicrobial response. We link these phenomena to two O. tsutsugamushi effectors that bind importin β1 and exploit its nuclear import function to enter the nucleus, inhibit NF-κB-dependent transcriptional activation, interact with exportin 1 and p65, and co-opt exportin 1 activity to mediate p65 nuclear export. We evidence the first examples of microbial proteins that exploit both importin and exportin activities to modulate NF-κB nuclear transport. As NF-κB is also associated with inflammation, autoimmunity, and cancer, dissecting how microbial proteins modulate NF-κB could not only lead to better understanding of infectious disease processes, but also could yield information that can ultimately be exploited to treat disorders caused by NF-κB dysregulation.

Salmonella enterica serovar Typhi, the causative agent of typhoid fever in humans, forms biofilms encapsulated by an extracellular matrix (ECM). Biofilms facilitate colonization and persistent infection in gallbladders of humans and mouse models of chronic carriage. Individual roles of matrix components have not been completely elucidated in vitro or in vivo . To examine individual functions, strains of Salmonella enterica serovar Typhimurium, the murine model of S . Typhi, in which various ECM genes were deleted or added, were created to examine biofilm formation, colonization, and persistence in the gallbladder. Studies show that curli contributes most significantly to biofilm formation. Expression of Vi antigen decreased biofilm formation in vitro and virulence and bacterial survival in vivo without altering the examined gallbladder pro- or anti-inflammatory cytokines. Oppositely, loss of all ECM components (Δ wcaM Δ csgA Δ yihO Δ bcsE ) increased virulence and bacterial survival in vivo and reduced gallbladder interleukin-10 (IL-10) levels. Colanic acid and curli mutants had the largest defects in biofilm-forming ability and contributed most significantly to the virulence increase of the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant strain. While the Δ wcaM Δ csgA Δ yihO Δ bcsE mutant was not altered in resistance to complement or growth in macrophages, it attached and invaded macrophages better than the wild-type (WT) strain. These data suggest that ECM components have various levels of importance in biofilm formation and gallbladder colonization and that the ECM diminishes disseminated disease in our model, perhaps by reducing cell attachment/invasion and dampening inflammation by maintaining/inducing IL-10 production. Understanding how ECM components aid acute disease and persistence could lead to improvements in therapeutic treatment of typhoid fever patients.

The intracellular bacterial pathogenSalmonellais able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous,Salmonella-specific CD4⁺helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen’s ability to persistin vivo.

Orientia tsutsugamushi is an obligate intracellular bacterium that infects mononuclear and endothelial cells to cause the emerging global health threat, scrub typhus. The ability of O. tsutsugamushi to survive in monocytes facilitates bacterial dissemination to endothelial cells, which can subsequently lead to several potentially fatal sequelae. As a strict intracellular pathogen that lives in the cytoplasm of host cells, O. tsutsugamushi has evolved to counter adaptive immunity. How the pathogen does so and outcome of this strategy in monocytes versus endothelial cells is poorly understood. This report demonstrates that O. tsutsugamushi reduces cellular levels of NOD-, LRR-, and CARD-containing 5 (NLRC5), a recently identified specific transactivator of major histocompatibility complex class-I (MHC-I) component gene expression, to inhibit MHC-I biosynthesis. Importantly, the efficacy of this approach varies with the host cell type infected. In non-professional antigen presenting HeLa and primary human aortic endothelial cells, O. tsutsugamushi-mediated reduction of NLRC5 results in lowered MHC-I component transcription and consequently less total and/or surface MHC-I levels throughout 72 h of infection. However, in infected THP-1 monocytes, which are professional antigen-presenting cells, reductions in NLRC5 and MHC-I observed during the first 24 h reverse thereafter. O. tsutsugamushi is the first example of a microbe that targets NLRC5 to modulate the MHC-I pathway. The differential ability of O. tsutsugamushi to modulate this pathway in non-professional versus professional antigen presenting cells could influence the morbidity and mortality of scrub typhus.