Interaction of Shigella flexneri IpaC with model membranes correlates with effects on cultured cells

Invasion of enterocytes by Shigella flexneri requires the properly timed release of IpaB and IpaC at the host-pathogen interface; however, only IpaC has been found to possess quantifiable activities in vitro. We demonstrate here that when added to cultured cells, purified IpaC elicits cytoskeletal changes similar to those that occur during Shigella invasion. This IpaC effect may correlate with its ability to interact with model membranes at physiological pH and to promote entry by an ipaC mutant of S. flexneri.     

The prevalence of herbal medicine home use and concomitant use with pharmaceutical medicines in Jamaica

Ethnopharmacological relevance

The work described in this paper aimed to study the prevalence of herbal medicine use in treating illness and concomitant use with pharmaceutical medicines in Jamaica.

Materials and methods

A survey using a structured questionnaire was administered by a trained interviewer to randomly selected adults in systematically selected households within randomly selected urban and rural clusters. Categorical data analysis was performed using Stata version 10 software.

Results

91.4% (372/407) of selected people agreed to participate. 72.6% (270/372) self-medicated with herbs within the previous year. Commonly treated were illnesses of the respiratory system (RS, 77.8% (210/270)), gastro-intestinal tract (GIT, 53.3% (144/270)) and health maintenance using tonics (29.6% (80/270)). 26.7% (72/270) of respondents used pharmaceuticals concomitantly with medicinal plants. Commonly treated were illnesses of the RS (20.4% (55/270)), GIT (13.7% (37/270)) and hypertension (10.0%(27/270)). 19.4% (14/72) of physicians knew of such practices. There was significant association of herb use with/without drugs with age (p < 0.001), employment status (p < 0.001), religion (p = 0.004), gender (p = 0.02) and educational level (p = 0.031). Thus prevalence of herb use alone was greatest amongst people aged 35-44 and 45-54 years; those employed; Rastafarians; those without health insurance; males and people who had completed secondary education. Whilst prevalence of concomitant herb-drug use was greater amongst people aged 65 years and older; those retired; those of religions other than Rastafarians and Christians, females and people who had attained primary education and below.

Conclusions

Self-medication with herbs in Jamaica is highly prevalent and highest for self-limiting conditions of the RS, GIT and health maintenance with tonics. Concomitant herb and drug use is highest for self-limiting conditions of the RS, GIT and hypertension, and the use of combined therapy highlights the need for investigations on potential drug-herb interactions. Physicians have limited awareness and knowledge of such concomitant usage, further highlighting the need for increased dialogue with patients, knowledge of medicinal plants and their uses and a heightened pharmacovigilance to avoid adversities that may arise from potential drug-herb interactions.     

Antigen-specific B memory cell responses to lipopolysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in volunteers vaccinated with live-attenuated Shigella flexneri 2a vaccine candidates

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B_M cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10⁶ expanded cells following vaccination (p = 0.008). A strong correlation was found between post-vaccination anti-LPS B_M cell counts and peak serum anti-LPS IgG titers (rs = 0.95, p = 0.0003). Increases in B_M specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B_M cells to LPS and IpaB, suggesting that B_M responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.     

Impact of Detergent on Biophysical Properties and Immune Response of the IpaDB Fusion Protein,a Candidate Subunit Vaccine against Shigella Species

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB''s cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55°C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90°C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine.     

Antibody response of monkeys to invasion plasmid antigen D after infection with Shigella spp.

The antigen preparation most often used for determining the levels of antibodies to virulence-associated proteins of Shigella spp. consists of a mixture of proteins (including IpaB, IpaC, IpaD, and VirG*) extracted from virulent shigellae with water (water extract). To overcome the lack of specificity for individual antigens in the water-extract enzyme-linked immunosorbent assay (ELISA), the ipaD gene from S. flexneri has been cloned, expressed to a high level, and purified for use in a new ELISA for the determination of the levels of antibody against IpaD in monkeys and humans challenged with shigellae. The IpaD ELISA for serum immunoglobulins G and A correlated well with the water-extract ELISA in that monkeys infected with S. flexneri or S. sonnei responded with high serum antibody titers in both assays. The IpaD assay required less antigen per well, had much lower background levels, and did not require correction with antigens from an avirulent organism. In conjunction with the water-extract ELISA, it was possible to identify infected animals that did not respond to IpaD but did produce antibodies that reacted in the water-extract ELISA. This indicates that even though IpaB, IpaC, and IpaD are essential for the invasiveness phenotype, the infected host does not always produce antibodies against all components of the invasiveness apparatus.     

Broadly protective Shigella vaccine based on type III secretion apparatus proteins

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.     

IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri

Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.     

Phase I evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral Delta virG Shigella sonnei vaccine candidate. In a double-blind, randomized, dose-escalating fashion, inpatient volunteers received a single dose of either placebo (n = 7) or vaccine (n = 27) at 3 x 10(3) CFU (group 1), 3 x 10(4) CFU (group 2), 3 x 10(5) CFU (group 3), or 3 x 10(6) CFU (group 4). The vaccine was generally well tolerated, although a low-grade fever or mild diarrhea occurred in six (22%) of the vaccine recipients. WRSS1 was recovered from the stools of 50 to 100% of the vaccinees in each group. The geometric mean peak anti-lipopolysaccharide responses in groups 1 to 4, respectively, were 99, 39, 278, and 233 for immunoglobulin (IgA) antibody-secreting cell counts; 401, 201, 533, and 284 for serum reciprocal IgG titers; and 25, 3, 489, and 1,092 for fecal IgA reciprocal titers. Postvaccination increases in gamma interferon production in response to Shigella antigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 10(3) to 10(6) CFU but elicits clinical reactions that must be assessed in further volunteer trials.     

Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion.

Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells.     

Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme.

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.