The rfaH gene, which affects lipopolysaccharide synthesis in Salmonella enterica serovar Typhi, is differentially expressed during the bacterial growth phase.
ABSTRACT We have cloned and sequenced the rfaH gene from Salmonella enterica serovar Typhi strain Ty2. The gene showed a high degree of similarity to the rfaH genes from Escherichia coli K-12 and S. enterica serovar Typhimurium. A rfaH mutant was constructed by site-directed mutagenesis. This mutant produced a rough lipopolysaccharide (LPS), with an incomplete core region. The defect in LPS expression that results from the rfaH mutation was corrected by a plasmid carrying the intact gene. The plasmid-borne rfaH gene also restored normal LPS synthesis in a rfaH mutant of E. coli. Reverse transcription-polymerase chain reaction analyses were performed to determine the effects of various environmental conditions on the expression of rfaH. The transcription of rfaH showed a growth-phase-dependent regulation, with maximal expression at the late exponential phase. Other environmental conditions, such as temperature or medium osmolarity, did not affect transcription of rfaH.
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ABSTRACT: RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A DeltarfaH mutant strain is attenuated in mice (50% lethal dose [LD(50)], >10(8) CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC P(BAD) promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the P(BAD) promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, DeltaP(rfaH178), was introduced into the attenuated vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) expressing the pneumococcal surface protein PspA from an Asd(+) balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic DeltarfaH derivative or the isogenic RfaH(+) parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD(50) of virulent Streptococcus pneumoniae WU2. Immunization with DeltaP(rfaH178) mutant strains led to increased levels of protection compared to that of the parent chi9241 and of a DeltarfaH derivative of chi9241.Infection and immunity 10/2009; 77(12):5572-82. · 4.21 Impact Factor
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ABSTRACT: Efficacious vaccination needs to confer protection against the vast majority of pathogens capable of causing a particular disease. Development of such vaccines is hindered by the great variability of microbes. Most pathogens have evolved variants that are able to express non-uniform surface structures. Naturally, evolutionary pressure has selected the most immunogenic antigens to be the most versatile. A combination of these multiform surface antigens forms the basis of classification of microbes into serotypes. Unfortunately, immune response in most cases is serotype-dependent, i.e. cross-protection among serotypes/serogroups of a given pathogen is limited. This review focuses on the strategies used for the engineering of broad-protective vaccine candidates, i.e., vaccines that induce a global, serotype-independent protection. The most plausible approach is to immunize with a multivalent vaccine containing different serotypes or purified serotype-determining antigens of a given pathogen. This arrangement is, however, efficient only against those microbes that have a limited number of serotypes, or few serotypes are responsible for the majority of the infections. Instead of using multivalent vaccine cocktails, cross-protective capacity of vaccine strains could be improved by making the conserved (i.e., shared by all variants) antigens more immunogenic. Elimination or down-regulation of the non-uniform antigens may increase immunogenicity of conserved minor antigens in vaccine candidates. Alternatively, shared antigens might be over-expressed in homologous or heterologous attenuated strains. Finally, purified conserved antigens could be used as subunit vaccines. In this paper, advantages and drawbacks of several such approaches will be reviewed.International journal of medical microbiology: IJMM 08/2008; 298(5-6):379-95. · 4.54 Impact Factor
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ABSTRACT: The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.International journal of medical microbiology: IJMM 09/2008; 299(2):109-19. · 4.54 Impact Factor