Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

ABSTRACTWe previously showed thatBrucella abortusrough mutant strain 2308 ΔATP(called the ΔrfbEmutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbEmutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbEmutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation ofasp24occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein generfbE(bab1_0542) and the permease generfbD(bab1_0543), while the ΔwboArough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes inasp24expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbEmutant by deleting thewboAgene (thereby creating the ΔrfbEΔwboAdouble-knockout strain) recoveredasp24expression. These results indicated thatasp24upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated thatasp24upregulation in the ΔrfbEmutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of theasp24gene favors intracellular survival ofBrucellain RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism forasp24upregulation inB. abortusmutants.

In VivoDifferences in the Virulence, Pathogenicity, and Induced Protective Immunity ofwboAMutants from Genetically Different Parent Brucella spp.

ABSTRACTTo explore the effects of the genetic background on the characteristics ofwboAgene deletion rough mutants generated from different parentBrucellasp. strains, we constructed the rough-mutant strainsBrucella melitensis16 M-MB6,B. abortus2308-SB6,B. abortusS19-RB6, andB. melitensisNI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks forB. abortusS19-RB6 to 11 weeks forB. abortus2308-SB6 andB. melitensisNI-NB6. However,B. abortusS19-RB6 andB. melitensis16 M-MB6, with a shorter survival time in mice, offered better protection against challenges withB. abortus2308 in protection tests thanB. abortus2308-SB6 andB. melitensisNI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival timein vivo. In the cross-protection assay, bothB. melitensis16 M-MB6 andB. abortusS19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated withB. abortusS19-RB6 andB. melitensis16 M-MB6,B. abortusS19-RB6 did not induce abortion, whereasB. melitensis16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunityin vivoin thewboAdeletion mutants from genetically different parentBrucellaspp. and also indicated that future rough vaccine strain development could be promising if suitable parentBrucellastrains and/or genes were selected.

Plasma lipopolysaccharide level and enterocyte brush border enzymes in gnotobiotic piglets infected with Salmonella typhimurium

Gnotobiotic piglets were orally infected either with the virulent LT2 strain or the non-pathogenic SF1591 rough mutant of Salmonella enterica serotype Typhimurium. They were sacrificed 6 or 24 h after the infection. All piglets infected for 24 h developed systemic infection with an increase of plasma lipopolysaccharide. Infection with the virulent strain caused a significant decrease (P < 0.001) of gamma-glutamyl transpeptidase (GGT) activity in the enterocyte brush border of both the jejunum and ileum, infection with the rough mutant caused a decrease of GGT activity in the ileum only. The activities of other brush border enzymes (lactase, sucrase, glucoamylase, alkaline phosphatase and dipeptidylpeptidase IV) did not change significantly after infection.