Microbiology and Immunology

Gu, Wenpeng; Wang, Xin; Qiu, Haiyan; Luo, Xia; Xiao, Di; Xiao, Yuchun; Tang, Liuying; Kan, Biao; Jing, Huaiqi

doi: 10.1111/j.1348-0421.2012.00478.xpmid: 22671974

Yersinia enterocolitica is a Gram‐negative enteric pathogen responsible for a number of gastrointestinal disorders; the most pathogenic bio‐serotype is 1B/O: 8. In this study, we compared the antigenicity of the outer membrane proteins and proteomics of the whole‐cell proteins of a pathogenic bio‐serotype 2/O: 9 isolated in China and a bio‐serotype 1B/O: 8 strain isolated in Japan. Using two‐dimensional gel electrophoresis, we showed that the outer membrane proteins A (OmpA), C (OmpC) and F (OmpF) were the major antigens for both strains, although proteins located on the bacterial cell membrane and enzymes involved in energy metabolism were also identified as antigenic. We compared the whole‐cell proteins of the two strains cultured at 25°C and 37°C and found portions of the outer membrane proteins (OmpX, OmpF and OmpA) were downregulated when the bacteria were cultured at 37°C, whereas urease subunit gamma (UreA), urease subunit alpha (UreC) and urease accessory protein (UreE), which are involved in urease synthesis, were upregulated when the bacteria were grown at 37°C. These observations will lay a foundation to selection of diagnostic markers for pathogenic Yersinia enterocolitica, and maybe contribute to choose the vaccine targets.

Jang, Jung Im; Kim, Jin Seok; Eom, Jeong Seon; Kim, Hyeon Guk; Kim, Bae Hoon; Lim, Sangyong; Bang, Iel‐Soo; Park, Yong Keun

doi: 10.1111/j.1348-0421.2012.00480.xpmid: 22708880

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N‐terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non‐toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N‐terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC‐specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N‐terminus of SipB enhance the induction of antigen‐specific immune responses, and that the N‐terminal domain of SipB can be used as a versatile delivery system for foreign antigens.

Nagano, Tetsuji; Kitahara, Rie; Nagai, Shinya

doi: 10.1111/j.1348-0421.2012.00482.xpmid: 22708916

Here construction of an attenuated mutant of an avian pathogenic Escherichia coli serovar O78 using an allelic exchange procedure is described. The mutant AESN1331, which carries a deletion in the crp gene, lost tryptophan deaminase activity and therefore lacked the ability to produce indole. The mutant strain additionally lacked the ability to adsorb Congo red, no longer fermented sugars other than glucose and L‐arabinose, did not harbor four known virulence‐associated genes (iss, tsh, cvaA, papC), and was susceptible to many antimicrobials, with the exception of nalidixic acid. The lethal dose (LD50 value) of the mutant strain on intravenous challenge in chickens was approximately 10‐fold higher than that of the parent strain. Additionally, the mutant strain was rapidly eliminated from chickens, being detected in the respiratory tract only on the first day post‐inoculation by fine spray. Administration of the mutant strain via various routes such as spray and eye drop for chickens, as well as in ovo inoculation for embryonated egg, evoked an effective immune response that protected against a virulent wild‐type E. coli O78 strain. Specifically, after immunization with the mutant strain, chickens challenged intravenously with an E. coli O78 strain exhibited decreases in mortality, clinical scores, organ lesion scores, and recovery of the challenge strain from organs compared to non‐immunized chickens. These findings suggest that AESN1331 is a suitable candidate for a live vaccine strain to protect chickens from colibacillosis caused by avian E. coli O78.

Choi, Hyun Jong; Shin, Myeong Su; Lee, Sang Myeong; Lee, Wan Kyu

doi: 10.1111/j.1348-0421.2012.00486.xpmid: 22709265

The aim of this study was to evaluate the immunomodulatory properties of Enterococcus faecium JWS 833 (JWS 833) isolated from duck intestine and compare them to those of Lactobacillus rhamnosus GG (LGG), a proven immunity‐enhancing probiotic. To investigate the immune‐enhancing properties of JWS 833, production of nitric oxide (NO) and cytokines was measured in mouse peritoneal macrophages. In addition, a Listeria monocytogenes challenge model was used in the assessment. It was found that heat‐killed JWS 833 stimulates mouse peritoneal macrophages to produce NO, interleukin‐1 β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) and that oral administration of viable JWS833 enhances NO, IL‐1β and TNF‐α synthesis upon L. monocytogenes challenge. Moreover, mice fed with JWS 833 were partially protected against lethal challenge with L. monocytogenes. JWS 833 strain has significantly greater immunostimulatory properties than LGG. Moreover, JWS 833 strain partially protects mice against lethal challenge with L. monocytogenes. JWS 833, a novel strain of E. faecium isolated from duck intestine, is potentially a useful feed supplement for controlling pathogens and enhancing host immune responses.

Yadav, Mukesh Kumar; Kwon, Seong Keun; Cho, Chang Gun; Park, Seok‐Won; Chae, Sung‐Won; Song, Jae‐Jun

doi: 10.1111/j.1348-0421.2012.00483.xpmid: 22708961

In this study, the gene expression profile of early in vitro Streptococcus pneumoniae biofilm with respect to planktonic cells in cDNA microarray analysis is reported. Microarray analysis with respect to planktonic cells was performed on total RNA extracted from biofilms grown in 24‐well microtiter plates. To validate the microarray results, real‐time RT‐PCR was performed on 13 differentially expressed genes and one constitutively expressed gene. The cDNA‐microarray analyses identified 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Genes involved in isoprenoid biosynthesis, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways were exclusively expressed in the biofilms, whereas transcription regulator genes were exclusively expressed in planktonic cells. The real‐time RT‐PCR results of 13 differentially regulated genes were completely in agreement with the microarray data. The exclusive up regulation in biofilms of genes involved in the mevalonate pathway, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways suggests that expression of these genes may be required for initial biofilm formation, and growth and survival of bacteria in biofilms. The up regulation of related genes suggests that cells in biofilms may be under stress conditions and possibly actively involved in the protein synthesis required to adapt to a new environment.

Fujii, Yoshiki; Shimoike, Takashi; Takagi, Hirotaka; Murakami, Kosuke; Todaka‐Takai, Reiko; Park, YoungBin; Katayama, Kazuhiko

doi: 10.1111/j.1348-0421.2012.00479.xpmid: 22708835

Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double‐stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full‐length sequences of long RNA segments of rotaviruses by one‐time only RT‐PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high‐fidelity and rapid DNA polymerase in a one‐step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS‐1, Hochi, 69M, WI61, M37 and SA11‐S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.

Suzuki, Tadaki; Semba, Shingo; Sunden, Yuji; Orba, Yasuko; Kobayashi, Shintaro; Nagashima, Kazuo; Kimura, Takashi; Hasegawa, Hideki; Sawa, Hirofumi

doi: 10.1111/j.1348-0421.2012.00484.xpmid: 22708997

JC virus (JCV) belongs to the polyomavirus family of double‐stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T′ antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein‐negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus‐like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S‐transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1‐pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.

Teodoro, Cristiane R. S.; Mattos, Cláudio S.; Cavalcante, Fernanda S.; Pereira, Eliezer M.; Santos, Kátia R. N. dos

doi: 10.1111/j.1348-0421.2012.00481.xpmid: 22672011

This work characterizes MLSb resistance in 39 methicillin‐resistant Staphylococcus aureus (MRSA) and 32 Staphylococcus epidermidis (MRSE) isolates. Of 21 erm(A) gene encoding MRSA isolates, 71.4% carried SCCmecIII, whereas of 12 isolates carrying the erm(C) gene, 83.3% carried SCCmecIV. Among the 25 MRSE isolates positive for the erm(C) gene, 80% had SCCmecIV or nontypeable cassettes. Isolates carrying these genes had MIC90≥ 256 μg/mL to erythromycin and clindamycin. The msr(A) gene was associated with a low MIC90 to these drugs. The erm(A) gene was associated with SCCmecIII in MRSA isolates, whereas the erm(C) gene was associated with SCCmecIV in both MRSA and MRSE isolates.

Matsumoto, Yuji; Kawamura, Yoshiki; Nakai, Hidetaka; Sugata, Ken; Yoshikawa, Akiko; Ihira, Masaru; Ohashi, Masahiro; Kato, Tomochika; Yoshikawa, Tetsushi

doi: 10.1111/j.1348-0421.2012.00489.xpmid: 22734496