Alfredo Menendez

Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-Induced Colitis

ABSTRACT Antibiotics are often used in the clinic to treat bacterial infections, but the effects of these drugs on microbiota composition and on intestinal immunity are poorly understood. Citrobacter rodentium was used as a model enteric pathogen to investigate the effect of microbial perturbation on intestinal barriers and susceptibility to colitis. Streptomycin and metronidazole were used to induce alterations in the composition of the microbiota prior to infection with C. rodentium. Metronidazole pretreatment increased susceptibility to C. rodentium-induced colitis over that of untreated and streptomycin-pretreated mice, 6 days postinfection. Both antibiotic treatments altered microbial composition, without affecting total numbers, but metronidazole treatment resulted in a more dramatic change, including a reduced population of Porphyromonadaceae and increased numbers of lactobacilli. Disruption of the microbiota with metronidazole, but not streptomycin treatment, resulted in an increased inflammatory tone of the intestine characterized by increased bacterial stimulation of the epithelium, altered goblet cell function, and thinning of the inner mucus layer, suggesting a weakened mucosal barrier. This reduction in mucus thickness correlates with increased attachment of C. rodentium to the intestinal epithelium, contributing to the exacerbated severity of C. rodentium-induced colitis in metronidazole-pretreated mice. These results suggest that antibiotic perturbation of the microbiota can disrupt intestinal homeostasis and the integrity of intestinal defenses, which protect against invading pathogens and intestinal inflammation.

Identification and Characterization of a Peptide That Specifically Binds the Human, Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody b12

ABSTRACT Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.

Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever

The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterial pathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonella infect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicated within gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa. Infected gallbladders showed histopathological damage characterized by destruction of the epithelium and massive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damage was determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of high numbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establish gallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvement of these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever.

Impact of Salmonella Infection on Host Hormone Metabolism Revealed by Metabolomics

ABSTRACT The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.

A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

MAb 2G12 neutralizes HIV‐1 by binding with high affinity to a cluster of high‐mannose oligosac charides on the envelope glycoprotein, gp120. Screening of phage‐displayed peptide libraries with 2G12 identified peptides that bind specifically, with Kds ranging from 0.4 to 200 μM. The crystal structure of a 21‐mer peptide ligand in complex with 2G12 Fab was determined at 2.8 Å resolution. Comparison of this structure with previous structures of 2G12‐carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohy drate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate‐binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man9GlcNAc2 (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydro gen bonds in contrast to six with Man9GlcNAc2, and only three of the antibody residues that interact with Man9GlcNAc2 also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not sup port structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligo saccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since anti sera produced against it did not bind gp120.—Menendez, A., Calarese, D. A., Stanfield, R. L., Chow, K. C., Scanlan, C. N., Kunert, R., Katinger, H., Burton, D. R., Wilson, I. A., Scott, J. K. A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. FASEB J. 22, 1380–1392 (2008)

The Deubiquitinase Activity of the Salmonella Pathogenicity Island 2 Effector, SseL, Prevents Accumulation of Cellular Lipid Droplets

ABSTRACT To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

Metabolomics Reveals Phospholipids as Important Nutrient Sources during Salmonella Growth in Bile In Vitro and In Vivo

During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.

Broadly reactive antibodies against a gp120 V3 loop multi-epitope polypeptide neutralize different isolates of human immunodeficiency virus type 1 (HIV-1).

A gene encoding for a novel multi-epitope polypeptide (TAB4) was synthesized and expressed in Escherichia coli. The protein was composed of 15 amino acid fragments derived from the V3 loop of HIV-1 isolates MN, IIIB, RF, JY1, BRVA and LR150, joined by five-amino-acid linkers. Immunogenicity of TAB4 in rabbits was studied, and the antibody response against individual peptides investigated. TAB4 was shown to be immunogenic in Complete Freunds Adjuvant in a dose-dependent manner, and was able to elicit a humoral response against all V3 epitopes included on the protein. Sera from some of the animals were able to neutralize the replication of viral strain MN, and in one case IIIB, with moderate titers. Some sera also neutralized several Cuban clinical strains, isolated in peripheral blood mononuclear cells, after one round of amplification in MT4 cells.

Defensins keep the peace too

The mammalian intestine contains a large number of commensal bacterial strains. New work suggests that antimicrobial peptides used for defense against pathogenic bacteria are also used to adjust the balance among bacterial populations and to control intestinal homeostasis.

NLRC5 elicits antitumor immunity by enhancing processing and presentation of tumor antigens to CD8+ T lymphocytes

ABSTRACT Cancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025–33 to Pmel-1 TCR transgenic CD8+ T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8+ T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.