Effect of Human Immunodeficiency Virus Infection on Plasma Bactericidal Activity against Salmonella enterica Serovar Typhimurium

Individuals with human immunodeficiency virus (HIV) infection have increased susceptibility to invasive disease caused by Salmonella enterica serovar Typhimurium. Studies from Africa have suggested that this susceptibility is related in part to the development of a high level of lipopolysaccharide (LPS)-specific IgG that is able to inhibit the killing of S. Typhimurium by bactericidal antibodies in healthy individuals. To explore this issue further, we examined the bactericidal activity against S. Typhimurium using serum and plasma samples from healthy controls and various clinical subgroups of HIV-infected adults in the United States. We found that the bactericidal activity in the samples from HIV-positive elite controllers was comparable to that from healthy individuals, whereas it was significantly reduced in HIV-positive viremic controllers and untreated chronic progressors. As demonstrated previously for healthy controls, the bactericidal activity of the plasma from the elite controllers was inhibited by preincubation with S. Typhimurium LPS, suggesting that it was mediated by anti-LPS antibodies. S. Typhimurium LPS-specific IgG was significantly reduced in all subgroups of HIV-infected individuals. Interestingly, and in contrast to the healthy controls, plasma from all HIV-positive subgroups inhibited in vitro killing of S. Typhimurium by plasma from a healthy individual. Our results, together with the findings from Africa, suggest that multiple mechanisms may be involved in the HIV-induced dysregulation of humoral immunity to S. Typhimurium.     

Design of Glycoconjugate Vaccines against Invasive African Salmonella enterica Serovar Typhimurium

Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S. Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM₁₉₇). The OAg-CRM₁₉₇ conjugates varied in (i) OAg source, with three S. Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM₁₉₇ ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S. Typhimurium glycoconjugate by challenge with live Salmonella. We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM₁₉₇ ratio. In addition, we found that bactericidal activity can be influenced by S. Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S. Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S. Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.     

Role of Salmonella Pathogenicity Island 1 Protein IacP in Salmonella enterica Serovar Typhimurium Pathogenesis

Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.The injection of bacterial proteins by the type III secretion system (T3SS) into the host cytoplasm has been broadly applied to study pathogen-host interactions ranging from the invasion of plant and animal pathogens to a symbiont interaction of Rhizobium (22, 42). The T3SS is composed of more than 20 different structural proteins that form needle-like appendages through which effector proteins are delivered directly into host cells to manipulate various host cell signaling events. Moreover, cytoplasmic chaperones are involved in the stability and efficient translocation of effector proteins (14). Salmonella enterica serovar Typhimurium, a facultative intracellular pathogen, has evolved two distinct T3SSs encoded by Salmonella pathogenicity island 1 (SPI-1), responsible for the invasion of nonphagocytic cells, and by SPI-2, required for intracellular survival and replication inside the Salmonella-containing vacuole (SCV). The expressions of the two T3SSs are inversely regulated during the pathogenic process. Although the expression of the SPI-1 T3SS at systemic sites has remained controversial, some effector proteins of SPI-1 (e.g., SipA and SopB) are persistently expressed and secreted under favorable conditions for SPI-2 expression during the biogenesis and maturation of the SCV (17).After the SPI-1 T3SS is activated upon host cell contact, the translocators SipB and SipC appear to be inserted into the host cell membrane, where they form a translocation pore, which is connected to the needle complex. A variety of effector proteins encoded within and outside SPI-1 can be translocated into a host cytoplasm and cooperatively induce membrane ruffling (11) and macropinocytosis (16). Among SPI-1 effector proteins, SopE, SopE2, and SopB trigger the actin rearrangement in host cells by activating small GTPases, including Rac1, Cdc42, and RhoG, directly or indirectly (39). A Salmonella serovar Typhimurium mutant carrying null mutations in these effector proteins failed to invade epithelial cells. After bacterial invasion, an activated membrane was subsequently recovered by SptP, another effector protein possessing GTPase-activating protein activity (13).The iacP gene, which is located downstream of sicA- sipBCDA in the SPI-1 locus, was initially identified as a putative acyl carrier protein (ACP) by sequence similarity (26). ACP is an abundant small acidic and highly conserved protein that is essential for various biosynthetic pathways (5). In the process of fatty acid (FA) biosynthesis in Escherichia coli, ACP sequentially delivers the acyl intermediates for FA elongation as a cofactor of FA synthase (20). For the enzymatic activity of ACP, a prosthetic group 4′-phosphopantetheine (4′-PP) that was covalently incorporated into apo-ACP serves as the binding site of acyl groups. It was reported previously that the substitution of serine 36 in Escherichia coli ACP eliminated the attachment site of the 4′-PP and inhibited FA incorporation (27).In addition to lipid biosynthesis, acyl-ACP is required for various bacterial virulence processes: the synthesis of the lipid A moiety of lipopolysaccharide (LPS) (43) and the N-acylhomoserine lactones as signal molecules in quorum sensing (52) and the posttranslational modification of bacterial toxins such as E. coli hemolysin (HlyA) (24). The activation of HlyA requires posttranslational acylation at two internal lysine residues by ACP and the acyl transferase HlyC. The conformation of acylated HlyA is matured into a molten globular form comprised of disordered regions, which is necessary for the hemolytic effects of a toxin to occur (21).As a Salmonella serovar Typhimurium mutant that lacks an entire SPI-1 locus was found to grow as well as the wild type, it is predicted that IacP would be responsible for the modification of other proteins in the T3SS (26). However, it is not known which proteins are targeted by IacP or how the invasion process during SPI-1 activation is affected in the iacP mutant. In this study, we report that IacP promotes SopB, SopA, and SopD secretion during cell entry, thus contributing to the virulence of Salmonella serovar Typhimurium.     

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.     

Characterization of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated in Japan

A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.     

Genetic Analysis of Non-Hydrogen Sulfide-Producing Salmonella enterica Serovar Typhimurium and S. enterica Serovar Infantis Isolates in Japan

Whole-genome sequencing of non-H₂S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 β-lactamase. The lack of production of H₂S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.     

Characterization of the Murine T-Lymphocyte Response to Salmonella enterica Serovar Typhimurium Infection

Infection of mice with Salmonella enterica serotype Typhimurium induces a strong Th1 cell response that is central for the control of infection. We infected mice of a resistant background with a virulent strain of S. enterica serovar Typhimurium and analyzed the kinetics and magnitude of the T-cell response. After infection, the majority of CD4(+) and CD8(+) splenocytes acquired an activated phenotype, as indicated by expression levels of CD44 and CD62L. In addition, after 3 to 4 weeks of infection, more than 20% of the CD4(+) and more than 30% of the CD8(+) T cells produced gamma interferon (IFN-gamma) in response to short-term polyclonal stimulation. In contrast, we detected only a moderate (two- to threefold) expansion of both T-cell populations, and BrdU incorporation revealed that there was either no or only a limited increase in the in vivo proliferation of CD4(+) and CD8(+) T cells, respectively. Our results indicate that although an unexpectedly large population of both CD4(+) and CD8(+) T cells is activated and acquires the potential to secrete IFN-gamma, this activation is not paralleled by substantial expansion of these T-cell populations.     

Flagellin from Recombinant Attenuated Salmonella enterica Serovar Typhimurium Reveals a Fundamental Role in Chicken Innate Immunity

Recombinant attenuated Salmonella vaccines have been extensively studied, with a focus on eliciting specific immune responses against foreign antigens. However, very little is known about the innate immune responses, particularly the role of flagellin, in the induction of innate immunity triggered by recombinant attenuated Salmonella in chickens. In the present report, we describe two Salmonella enterica serovar Typhimurium vaccine strains, wild-type (WT) or flagellin-deficient (flhD) Salmonella, both expressing the fusion protein (F) gene of Newcastle disease virus. We examined the bacterial load and spatiotemporal kinetics of expression of inflammatory cytokine, chemokine, and Toll-like receptor 5 (TLR5) genes in the cecum, spleen, liver, and heterophils following oral immunization of chickens with the two Salmonella strains. The flhD mutant exhibited an enhanced ability to establish systemic infection compared to the WT. In contrast, the WT strain induced higher levels of interleukin-1β (IL-1β), CXCLi2, and TLR5 mRNAs in cecum, the spleen, and the heterophils than the flhD mutant at different times postinfection. Collectively, the present data reveal a fundamental role of flagellin in the innate immune responses induced by recombinant attenuated Salmonella vaccines in chickens that should be considered for the rational design of novel vaccines for poultry.     

Salmonella enterica Serovar Typhimurium Response Involved in Attenuation of Pathogen Intracellular Proliferation

Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate. S. enterica serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate. phoP- and phoQ-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named igaA for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.     

Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.Recognition of microbes is a critical step in the initiation of the host immune response against infection. Indeed, detection of microbe-associated molecular patterns by germ line-encoded receptors such as Toll-like receptors (18) and Nod-like receptors (NLRs) (8) is an early event that leads to inflammatory responses through the production of cytokines and chemokines. Nod1 and Nod2 are cytosolic proteins of the NLR family that detect distinct substructures from bacterial peptidoglycan (8). Whereas Nod2 detects muramyl dipeptide (12, 16), a motif common to gram-negative and gram-positive bacteria, Nod1 senses meso-diaminopimelic acid-containing peptidoglycan (3, 11), which is more commonly found in gram-negative bacteria. In macrophages and dendritic cells (DC), triggering of Nod1 and Nod2 induces proinflammatory cytokines and costimulatory molecules (21). In addition, synergistic effects of Nod ligands with Toll-like receptor ligands in myeloid cells have been reported (9, 29). Nod1 has been shown to regulate the colonization of mice by Helicobacter pylori (31), and Nod2 affects the pathogenicity of Listeria monocytogenes (19) and Mycobacterium tuberculosis (6) in mice models. However, no studies have been conducted on the impact of Nod1 and Nod2 on the in vivo infection process of the specific enteric pathogen, Salmonella enterica serovar Typhimurium.Salmonella enterica is a gram-negative bacterium of the Enterobacteriaceae family. S. enterica serovar Typhimurium is a mouse pathogen that provokes a typhoid-like syndrome in orally infected mice, with colonization of the deeper organs, including the liver and spleen (5). S. Typhimurium is capable of entering intestinal epithelial cells using a unique mechanism involving a type 3 secretion system, Salmonella pathogenicity island 1 (Spi1) (10), and resides in a vacuole within infected cells via a mechanism dependent on a second type 3 secretion system, Spi2 (27). Hence, the bacteria are able to avoid killing and spread throughout the infected host by invading immune cells. The intracellular lifestyle of Salmonella is in line with a possible implication of Nod proteins during the course of the infection. However, to date, no study has been conducted in vivo to determine the role of Nod1 and Nod2 after oral infection with S. Typhimurium.Spi1 is critical for the invasiveness of the bacteria in epithelial cells and is thought to be responsible for the main route of entry of the bacteria through Peyer''s patches (13, 30). Strikingly, bacteria deficient for Spi2 are completely avirulent whereas Spi1 mutants are still capable of inducing the disease (26). Recently, myeloid cells from the intestinal lamina propria have been shown to sample the luminal contents of the gut, including intact bacteria. This mechanism is crucial for gut homeostasis but provides a portal of entry for S. Typhimurium and explains the persistent virulence of Spi1-deficient bacteria (4, 24, 30).In the present study we show that Nod1 deficiency leads to increased susceptibility to Spi1 deficient-S. Typhimurium but not the wild-type (WT) strain, suggesting a critical role for Nod1 in myeloid cells from the intestinal lamina propria for defense against S. Typhimurium infection in vivo. Accordingly, using bone marrow-chimeric mice, we have been able to locate the defect in in vivo hematopoietic cells. Indeed, Nod1 deficient animals show increased S. Typhimurium in the lamina propria DC subpopulation and an impaired cellular response after infection with Spi1-deficient bacteria. Additionally, we observed an impaired response of Nod1-deficient DCs toward the bacteria. Taken together, our findings uncover a surprising role of Nod1 in lamina propria DCs in the control of S. Typhimurium infection in vivo.     

Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains

Salmonella enterica serovar Typhimurium is a versatile organism for the generation of live recombinant vaccines for mucosal immunization. Various strategies have been devised for the stable and efficient expression of heterologous antigens by attenuated S. enterica strains, but these methods often require complex manipulations. Use of phage lambda Red recombinase has recently been devised for gene replacements in Escherichia coli and S. enterica after introduction of PCR products. Based on this method, we have developed an approach that allows the integration of recombinant expression cassettes for heterologous antigens in a single step. The recombinant construct is integrated into the chromosome and is devoid of any selective marker such as antibiotic resistance. We observed the stable expression of model antigens without selective pressure. In addition, the method allows the simultaneous generation of attenuating mutations by gene deletions. The novel "knock-in" approach allows the rapid and efficient construction of recombinant Salmonella strains as vaccine carriers.     

Emergence and Characterization of Salmonella enterica Serovar Typhimurium Phage Type DT191a

The emergence of a previously undefined phage type of Salmonella enterica serovar Typhimurium, designated DT191a, occurred in England and Wales in July 2008. The new strain exhibits a number of distinctive phenotypic and genotypic features. This report provides the tools necessary to track S. Typhimurium DT191a globally.Salmonella enterica serovar Typhimurium is the second most prevalent serovar isolated in Europe, exceeded only by Salmonella enterica serovar Enteritidis (6). Provisional data for 2008 to July 2009 from the Health Protection Agency Salmonella data set show that S. Typhimurium accounted for 2,690 (18.8%) of Salmonella infections from humans in England and Wales. Subtyping of S. Typhimurium by phage typing is well established and not only plays a vital role in detecting outbreaks (11) but also enables the appearance of new phage types or phage types with new characteristics to be monitored.We report here on the emergence, in mid-2008, in England and Wales of a previously undefined phage type of S. Typhimurium and on the further characterization of this phage type by pulsed-field gel electrophoresis (PFGE), variable-number tandem repeat (VNTR) typing, and multilocus sequence typing (MLST). This strain was initially identified in our laboratory, as it did not conform to any of the recognized patterns in the current schemes as described by Callow (4) and Anderson et al. (1). Its unique pattern of phage lysis was subsequently defined as DT191a. The full phage reaction pattern with the panel of 32 typing phages is shown in Table ​Table1,1, where the only significant difference is in the reaction for phage 18. To date, over 230 cases of DT191a have been typed by the Laboratory of Gastrointestinal Pathogens (LGP), Health Protection Agency (HPA). In England and Wales, DT191a is currently the most common phage type of S. Typhimurium obtained from cases of human infection. The increased isolation rate of this phage type is compared with the level for DT104 in Fig. ​Fig.11.Open in a separate windowFIG. 1.Relative proportions of Salmonella enterica serovar Typhimurium DT104 and DT191a typed at the Salmonella Reference Laboratory, United Kingdom, between 2008 and 2009.

TABLE 1.

Phage reactions for the new phage type S. Typhimurium DT191a

PspA Family Fusion Proteins Delivered by Attenuated Salmonella enterica Serovar Typhimurium Extend and Enhance Protection against Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is highly immunogenic and can induce a protective immune response against pneumococcal infection. PspA is divided into two major families based on serological variability: family 1 and family 2. To provide broad protection, PspA proteins from pneumococcal strains Rx1 (family 1) and EF5668 (family 2) were combined to form two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. Each protein was fused to a type II secretion signal and delivered by a recombinant attenuated Salmonella vaccine (RASV). Both PspA/Rx1-EF5668 and PspA/EF5668-Rx1 were synthesized in the RASV and secreted into the periplasm and supernatant. The fusion proteins reacted strongly with both anti-PspA/Rx1 and anti-PspA/EF5668 antisera. Oral immunization of BALB/c mice with RASV synthesizing either PspA fusion protein elicited serum immunoglobulin G (IgG) and mucosal IgA responses against both families of PspA. Analysis of IgG isotypes (IgG2a and IgG1) indicated a strong Th1 bias to the immune responses to both proteins. Sera from mice immunized with RASV synthesizing PspA/Rx1-EF5668 bound to the surface and directed C3 complement deposition on representative strains from all five PspA clades. Immunization with RASV synthesizing either protein protected mice against intraperitoneal challenge with Streptococcus pneumoniae WU2 strain (family 1), intravenous challenge with S. pneumoniae 3JYP2670 strain (family 2), and intranasal challenge with S. pneumoniae A66.1 (family 1). The PspA/Rx1-EF5668 protein elicited significantly greater protection than PspA/EF5668-Rx1, PspA/Rx1, or PspA/EF5668. These results indicate an RASV synthesizing a PspA fusion protein representing both PspA families constitutes an effective antipneumococcal vaccine, extending and enhancing protection against multiple strains of S. pneumoniae.Streptococcus pneumoniae is a human pathogen causing significant morbidity and mortality worldwide, especially in developing countries. It causes respiratory infections, otitis media, sinusitis, and invasive diseases such as pneumonia, meningitis, and bacteremia. S. pneumoniae causes more than 1 million deaths worldwide every year among children under 5 years of age (8, 11, 20). The current 23-valent capsular polysaccharide vaccine elicits immunity in individuals greater than 2 years of age, and the current conjugate polysaccharide-protein pneumococcal vaccine provides protection for those under the age of 2 years (23, 26, 33). However, protection is restricted to only the limited number of serotypes included in the vaccine formulation (26), and the expensive production costs limit its use in developing countries. Moreover, serotype replacement has been observed in vaccinated populations and an increase in infections by pneumococcal serotypes not included in the 7-valent conjugated polysaccharide vaccine has been described recently (29, 56). In some countries, as many as 66% of childhood strains would not be covered (26, 45). Treatment of pneumococcal diseases has become more challenging due to the increase in multiple-drug-resistant pneumococcal strains (58). These issues reinforce the need for more affordable, broadly protective strategies for immunization against pneumococcal infection.Several pneumococcal proteins have been under investigation as potential vaccine candidates, including pneumococcal surface protein A (PspA) (7, 10, 14), pneumococcal surface protein C (PspC) (12), and pneumolysin (1, 50). PspA is a virulence factor expressed by all clinical S. pneumoniae isolates. It consists of five domains: (i) a signal peptide, (ii) an α-helical and charged domain that bears a strong 7-residue periodicity typical of coiled-coil proteins (amino acids [aa] 1 to 288), (iii) a proline-rich region (aa 289 to 370) which spans the cell wall and is highly conserved in all S. pneumoniae strains, (iv) a choline-binding domain consisting of 10 20-aa repeats (aa 371 to 571) that anchors the protein to the cell surface, and (v) a C-terminal 17-aa tail (aa 572 to 589) (Fig. ​(Fig.1).1). The α-helical region is variable in length and amino acid sequence, but the antibodies against this region are protective and cross-reactive. PspA proteins have been grouped into three families encompassing six different clades based on the C-terminal 100 aa of the α-helical region (28). Family 1 is comprised of clades 1 and 2, family 2 is comprised of clades 3, 4, and 5, and family 3 consists of clade 6. S. pneumoniae strains expressing family 1 or 2 PspA proteins constitute 98% of clinical isolates (27, 28, 53). To accommodate this variability, it was proposed that a combination of two PspA antigens, one from PspA family 1 and one from PspA family 2, would elicit protection against the vast majority of S. pneumoniae strains (27, 28, 47). In addition to the α-helical region, the proline-rich domain has been shown to encode protective epitopes (S. Hollingshead, unpublished observation). This region of the protein is highly conserved compared to the α-helical region, making inclusion of the proline-rich domain important to achieve broad protection (4, 9, 28).Open in a separate windowFIG. 1.Schematic diagram of PspA and PspA fusion proteins. At the top is the entire PspA molecule containing the N-terminal α-helical domain (region A), the proline-rich region (region B), the choline-binding domain (region C), and the C-terminal tail (region D). Each recombinant fusion protein is shown with its distinct domains.Complement-mediated opsonin-dependent phagocytosis is an important defense mechanism against pneumococcal infections. It activates both the classical and alternative complement pathways, depositing C3b on the pneumococcal surface (13, 34, 35). PspA inhibits complement activation (60), and anti-PspA antibodies can overcome this effect (53), leading to increased C3 deposition on the bacterial surface and enhanced clearance. Anti-PspA-directed C3 complement deposition has been correlated with protection against S. pneumoniae challenge in mice (19). Therefore, measurement of C3 complement deposition on the pneumococcal surface directed by sera from vaccinated individuals could be an important correlate of protection.Previous work in our laboratory demonstrated that recombinant avirulent Salmonella enterica serovar Typhimurium vaccines (RASVs) can be used to deliver PspA cloned from S. pneumoniae strain Rx1 (family 1) and induce protection in mice against challenge with homologous family 1 S. pneumoniae strain WU2 (38-40, 48, 64). Using RASV to deliver antigens has many advantages, including low-cost vaccine production, needle-free delivery, and induction of strong mucosal immunity (16, 18). In this article, gene fragments encoding the α-helix domain of PspA from family 1 strain Rx1 and the α-helix domain and proline-rich region of family 2 strain EF5668 were used to construct gene fusions encoding two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. These gene fusions were expressed and delivered by an RASV strain designed to regulate antigen expression by the availability of arabinose, resulting in regulated delayed antigen synthesis, to enhance and extend protection against S. pneumoniae clinical strains.     

Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections

Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. Anat Rec, 302:2224–2232, 2019. © 2019 American Association for Anatomy     

Diagnostic and public health dilemma of lactose-fermenting Salmonella enterica serotype Typhimurium in cattle in the Northeastern United States

The presence of lactose-fermenting Salmonella strains in clinical case materials presented to microbiology laboratories presents problems in detection and identification. Failure to detect these strains also presents a public health problem. The laboratory methods used in detecting lactose-fermenting Salmonella enterica serotype Typhimurium from six outbreaks of salmonellosis in veal calves are described. Each outbreak was caused by a multiply-resistant and lactose-fermenting strain of S. enterica serotype Typhimurium. The use of Levine eosin-methylene blue agar in combination with screening of suspect colonies for C8 esterase enzyme and inoculation of colonies into sulfide-indole-motility medium for hydrogen sulfide production was particularly effective for their detection. A hypothesis for the creation of lactose-fermenting salmonellae in the environment is presented. It is proposed that the environment and husbandry practices of veal-raising barns provide a unique niche in which lactose-fermenting salmonellae may arise.     

Defining the Core Genome of Salmonella enterica Serovar Typhimurium for Genomic Surveillance and Epidemiological Typing

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing foodborne infections in Australia and many other countries. Twenty-one S. Typhimurium strains from Salmonella reference collection A (SARA) were analyzed using Illumina high-throughput genome sequencing. Single nucleotide polymorphisms (SNPs) in 21 SARA strains ranged from 46 to 11,916 SNPs, with an average of 1,577 SNPs per strain. Together with 47 strains selected from publicly available S. Typhimurium genomes, the S. Typhimurium core genes (STCG) were determined. The STCG consist of 3,846 genes, a set that is much larger than that of the 2,882 Salmonella core genes (SCG) found previously. The STCG together with 1,576 core intergenic regions (IGRs) were defined as the S. Typhimurium core genome. Using 93 S. Typhimurium genomes from 13 epidemiologically confirmed community outbreaks, we demonstrated that typing based on the S. Typhimurium core genome (STCG plus core IGRs) provides superior resolution and higher discriminatory power than that based on SCG for outbreak investigation and molecular epidemiology of S. Typhimurium. STCG and STCG plus core IGR typing achieved 100% separation of all outbreaks compared to that of SCG typing, which failed to separate isolates from two outbreaks from background isolates. Defining the S. Typhimurium core genome allows standardization of genes/regions to be used for high-resolution epidemiological typing and genomic surveillance of S. Typhimurium.