Identification and characterization of a capsule in Shigella and its role in the pathogenesis

Shigella spp. are the etiologic agent of bacillary dysentery and one of the most frequent causes of diarrhea in developing countries, especially in children under five years of age. 50 different serotypes have been defined by the O antigen of the LPS. A key objective of the Novartis Vaccines Institute for Global Health is to develop a broadly-protective vaccine against Shigella based on outer membrane particles. Inactivation of OAg synthesis is of interest to avoid serotype-specific responses. Deletion of the gene galU results in OAg-deficiency in S. flexneri. Surprisingly, we found that immunization with Delta_galU S. sonnei 53G outer membrane particles or inactivated bacteria raised an OAg-specific response in mice. The presence of OAg on the surface of Delta_galU S. sonnei was confirmed by flow cytometry. GalU is involved in the synthesis of the LPS core but not in the synthesis of the OAg repeating units. A group 4 capsule (G4C) consisting of the same repeating units as the OAg side chain of LPS has been described in enteropathogenic E. coli. Shigella is reported to posses a homologous G4C operon. Size-Exclusion-HPLC analyses of exopolysaccharide extracts showed that S. sonnei WT and Delta_galU strains both expressed a polysaccharide with higher molecular weight than the predominant wild-type OAg chain of 20-25 units. NMR analysis identified the presence of the S. sonnei OAg in the high molecular weight polysaccharide (HMW-PS). The absence of this HMW-PS was confirmed by generating isogenic mutants in the G4C operon. Together, our results indicate for the first time that the G4C gene cluster is functional in S. sonnei and encodes the synthesis of a group 4 capsule and that this capsule is still present in the S. sonnei Delta_galU mutant. The function of the G4C as potential virulence factor of S. sonnei was investigated both in vitro and in vivo. Our results showed that the presence of the capsule polysaccharide accounted for changes in cell invasion ability both in vitro and in vivo. Moreover, the G4C modulated the inflammatory host responses during the infection and the abilities of bacteria to translocate and disseminate. In conclusion, this study describes a new and interesting finding in the Shigella field to achieve a better understanding of the pathogenic mechanism of Shigella and in addition of the function of extracellular polysaccharides as virulence factors. Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Therefore, the identification of a capsule in Shigella could have implications on the design of new-generation anti-Shigella drugs and also on the development of preventive measures, as a vaccine against this enteric disease

A Selective Antibiotic for Lyme Disease

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment

High Yield Production Process for Shigella Outer Membrane Particles

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30–45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from Gram-negative bacteria

Cranberry extracts promote growth of Bacteroidaceae and decrease abundance of Enterobacteriaceae in a human gut simulator model.

The opportunistic pathogen Escherichia coli, a common member of the human gut microbiota belonging to the Enterobacteriaceae family, is the causative agent of the majority of urinary tract infections (UTIs). The gut microbiota serves as a reservoir for uropathogenic E. coli where they are shed in feces, colonize the periurethral area, and infect the urinary tract. Currently, front line treatment for UTIs consists of oral antibiotics, but the rise of antibiotic resistance is leading to higher rates of recurrence, and antibiotics cause collateral damage to other members of the gut microbiota. It is commonly believed that incorporation of the American cranberry, Vaccinium macrocarpon, into the diet is useful for reducing recurrence of UTIs. We hypothesized such a benefit might be explained by a prebiotic or antimicrobial effect on the gut microbiota. As such, we tested cranberry extracts and whole cranberry powder on a human gut microbiome-derived community in a gut simulator and found that cranberry components broadly modulate the microbiota by reducing the abundance of Enterobacteriaceae and increasing the abundance of Bacteroidaceae. To identify the specific compounds responsible for this, we tested a panel of compounds isolated from cranberries for activity against E. coli, and found that salicylate exhibited antimicrobial activity against both laboratory E. coli and human UTI E. coli isolates. In a gut simulator, salicylate reduced levels of Enterobacteriaceae and elevated Bacteroidaceae in a dose dependent manner

A Distinct Microbiome Signature in Posttreatment Lyme Disease Patients.

Lyme disease is the most common vector-borne disease in the United States, with an estimated incidence of 300,000 infections annually. Antibiotic intervention cures Lyme disease in the majority of cases; however, 10 to 20% of patients develop posttreatment Lyme disease syndrome (PTLDS), a debilitating condition characterized by chronic fatigue, pain, and cognitive difficulties. The underlying mechanism responsible for PTLDS symptoms, as well as a reliable diagnostic tool, has remained elusive. We reasoned that the gut microbiome may play an important role in PTLDS given that the symptoms overlap considerably with conditions in which a dysbiotic microbiome has been observed, including mood, cognition, and autoimmune disorders. Analysis of sequencing data from a rigorously curated cohort of patients with PTLDS revealed a gut microbiome signature distinct from that of healthy control subjects, as well as from that of intensive care unit (ICU) patients. Notably, microbiome sequencing data alone were indicative of PTLDS, which presents a potential, novel diagnostic tool for PTLDS.IMPORTANCE Most patients with acute Lyme disease are cured with antibiotic intervention, but 10 to 20% endure debilitating symptoms such as fatigue, neurological complications, and myalgias after treatment, a condition known as posttreatment Lyme disease syndrome (PTLDS). The etiology of PTLDS is not understood, and objective diagnostic tools are lacking. PTLDS symptoms overlap several diseases in which patients exhibit alterations in their microbiome. We found that patients with PTLDS have a distinct microbiome signature, allowing for an accurate classification of over 80% of analyzed cases. The signature is characterized by an increase in Blautia, a decrease in Bacteroides, and other changes. Importantly, this signature supports the validity of PTLDS and is the first potential biological diagnostic tool for the disease

A Distinct Microbiome Signature in Posttreatment Lyme Disease Patients.

Lyme disease is the most common vector-borne disease in the United States, with an estimated incidence of 300,000 infections annually. Antibiotic intervention cures Lyme disease in the majority of cases; however, 10 to 20% of patients develop posttreatment Lyme disease syndrome (PTLDS), a debilitating condition characterized by chronic fatigue, pain, and cognitive difficulties. The underlying mechanism responsible for PTLDS symptoms, as well as a reliable diagnostic tool, has remained elusive. We reasoned that the gut microbiome may play an important role in PTLDS given that the symptoms overlap considerably with conditions in which a dysbiotic microbiome has been observed, including mood, cognition, and autoimmune disorders. Analysis of sequencing data from a rigorously curated cohort of patients with PTLDS revealed a gut microbiome signature distinct from that of healthy control subjects, as well as from that of intensive care unit (ICU) patients. Notably, microbiome sequencing data alone were indicative of PTLDS, which presents a potential, novel diagnostic tool for PTLDS.IMPORTANCE Most patients with acute Lyme disease are cured with antibiotic intervention, but 10 to 20% endure debilitating symptoms such as fatigue, neurological complications, and myalgias after treatment, a condition known as posttreatment Lyme disease syndrome (PTLDS). The etiology of PTLDS is not understood, and objective diagnostic tools are lacking. PTLDS symptoms overlap several diseases in which patients exhibit alterations in their microbiome. We found that patients with PTLDS have a distinct microbiome signature, allowing for an accurate classification of over 80% of analyzed cases. The signature is characterized by an increase in Blautia, a decrease in Bacteroides, and other changes. Importantly, this signature supports the validity of PTLDS and is the first potential biological diagnostic tool for the disease

Modulation of endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural environment and orientation. We previously developed a high yield production process for genetically derived particles, called Generalized Modules for Membrane Antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane and contain immune-stimulatory components, especially lipopolysaccharide (LPS), we examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes msbB or htrB in GMMA-producing S. sonnei and S. flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduction, GMMA from the S. sonnei ΔhtrB mutant a 60,000-fold reduced ability compared to GMMA with wild-type lipid A to stimulated human Toll-like receptor 4 (TLR4) in a reporter cell line. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory palmitoleoylation occurs resulting in hexa-acylated lipid A with approximately 10-fold higher activity than the penta-acylated lipid A. In human PBMC, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (800-fold for S. sonnei ΔhtrB, 300-fold for the msbB mutants) compared to a 50-fold reduction observed for GMMA with palmitoleoylated lipid A from the S. flexneri ΔhtrB strain. We demonstrated that the residual activity of GMMA with penta-acylated lipid A is largely due to non-lipid A related TLR2 activation whereas GMMA with palmitoleoylated hexa-acylated lipid A predominantly activate TLR4. These results identify the relative activation of TLR4 and TLR2 pathways by GMMA

An O Antigen Capsule Modulates Bacterial Pathogenesis in <i>Shigella sonnei</i>

<div><p><i>Shigella</i> is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. <i>S</i>. <i>flexneri</i> 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas <i>S</i>. <i>sonnei</i> LPS comprises a monomodal OAg. Here we reveal that <i>S</i>. <i>sonnei</i>, but not <i>S</i>. <i>flexneri</i> 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a <i>galU</i> mutant of <i>S</i>. <i>sonnei</i>, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a <i>galU</i> mutant of <i>S</i>. <i>flexneri</i> 2a cannot. High molecular weight material not linked to the LPS was purified from <i>S</i>. <i>sonnei</i> and confirmed by NMR to contain the specific sugars of the <i>S</i>. <i>sonnei</i> OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in <i>Escherichia coli</i>, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of <i>S</i>. <i>sonnei</i>. Uncapsulated knockout bacteria were highly invasive <i>in vitro</i> and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of <i>S</i>. <i>sonnei</i> to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness <i>in vitro</i> and inflammation <i>in vivo</i> compared to the wild type. In conclusion, the data indicate that in <i>S</i>. <i>sonnei</i> expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.</p></div