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: 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
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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: The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by Wzz(ST) and a very long (VL) OAg determined by Wzz(fepE). This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz(fepE) gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.Journal of Medical Microbiology 08/2008; 57(Pt 8):938-46. · 2.30 Impact Factor