ABSTRACT
Previous studies in our laboratory described a new group A streptococcal protective antigen (Spa) in type 18 streptococci that was distinct from the type 18 M protein. This study was undertaken to identify additional serotypes of group A streptococci that express Spa proteins. PCR techniques were used to identify and clone a new spa gene from type 36 streptococci. The 5' sequence of spa36 was highly variable compared to spa18, while the 3' sequence was conserved. Antisera against Spa36 opsonized type 36 streptococci but not type 18 streptococci, indicating that the opsonic Spa epitopes were type-specific. Antisera against the conserved carboxy-terminal half of Spa18 were used to identify Spa or Spa-like proteins expressed on the surface of 25 of 70 different serotypes of GAS. Spa proteins may represent a new family of type-specific surface antigens that function in concert with M proteins to elicit protective immune responses.
Subject(s)
Antigens, Bacterial/immunology , Opsonin Proteins/immunology , Phagocytosis , Streptococcus pyogenes/immunology , Animals , Antigens, Bacterial/genetics , Bacterial Typing Techniques , DNA Primers/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Disease Models, Animal , Humans , Mice , Mice, Inbred ICR , Molecular Sequence Data , Polymerase Chain Reaction/methods , Rabbits , Sequence Analysis, DNA , Serotyping , Streptococcal Infections/microbiology , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/classification , Streptococcus pyogenes/genetics , Survival AnalysisABSTRACT
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 sigma(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 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) 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 10(4) 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
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Infections/microbiology , Escherichia coli O157/pathogenicity , Feces/microbiology , Sigma Factor/metabolism , Animals , Bacterial Proteins/genetics , Cattle , Colony Count, Microbial , Escherichia coli O157/genetics , Escherichia coli O157/growth & development , Humans , Hydrogen-Ion Concentration , Mice , Mice, Inbred ICR , Mutation , Sigma Factor/geneticsABSTRACT
Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
