Fur-dependent detoxification of organic acids by rpoS mutants during prolonged incubation under aerobic, phosphate starvation conditions

The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (P_i) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, P_i starvation conditions. Whereas rpoS⁺ cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS (σ^S) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS⁺ cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and P_i excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.     

Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS⁺ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS⁺ vaccines induced a balanced Th1/Th2 immune response while the RpoS⁻ strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS⁺ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.     

BosR Functions as a Repressor of the ospAB Operon in Borrelia burgdorferi

The Lyme disease spirochete, Borrelia burgdorferi, must abundantly produce outer surface lipoprotein A (OspA) in the tick vector but downregulate OspA in mammals in order to evade the immune system and maintain its natural enzootic cycle. Here, we show that BosR binds two regulatory elements of the ospAB operon and that increasing BosR expression leads to downregulation of OspA. Both regulatory sequences, cisI and cisII, showed strong BosR-binding and cisII bound much tighter than cisI. A promoterless bosR gene fused with an inducible promoter was introduced into an rpoS mutant and a wild-type strain to assess RpoS-independent and -dependent downregulation of OspA by BosR. With the induction of BosR expression, OspA expression was reduced more significantly in the RpoS-deficient than wild-type background, but not completely repressed. In the presence of constitutive expression of OspC, DbpA and DbpB, increasing BosR production resulted in complete repression of OspA in the RpoS mutant. Taken together, the study clearly demonstrated BosR serves as a repressor that binds both regulatory elements of the ospAB operon and shuts off expression.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ς^S and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10¹ to 10⁴ CFU, we found the wild-type strain in feces of mice given lower doses (10² versus 10³ CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10⁴ CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ς^S appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.