Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection.

The cysteine protease of group A streptococci has been suggested to contribute to the pathogenesis of invasive infection through degradation of host tissue, activation of the host inflammatory response, release of protective molecules from the bacterial cell surface, or other mechanisms. However, studies of the effects on virulence of inactivating the cysteine protease gene speB have yielded conflicting results. In some reports, a speB mutant was relatively avirulent in mouse models of invasive infection whereas little or no attenuation of virulence was observed in other studies of similar mutant strains. Possible reasons for these discordant results include differences in the streptococcal strains from which the speB mutants were derived, differences in the infection models employed, or unintended effects on another virulence determinant(s) that arose during the derivation of a speB mutant. We attempted to clarify these issues by characterizing the phenotypic properties and relative virulence in mice of two speB mutant strains, both derived from wild-type strain AM3: speB mutant AM3speB, which has been shown to be markedly attenuated in virulence in mice after intraperitoneal or subcutaneous challenge, and AM3speBOmega, a new mutant strain derived for this investigation. Both mutant strains were negative for protease activity, as expected, and both produced wild-type amounts of type 3 M protein and streptolysin O. However, AM3speB produced significantly less cell-associated hyaluronic acid capsule than did parent strain AM3 or strain AM3speBOmega. Compared to wild-type strain AM3, AM3speB was more sensitive to opsonophagocytic killing in vitro and was significantly less virulent in mice after intraperitoneal challenge. By contrast, AM3speBOmega was fully resistant to phagocytosis and did not differ significantly from the wild-type strain in mouse virulence after an intraperitoneal or subcutaneous challenge. We concluded that previous reports attributing loss of virulence in strain AM3speB to inactivation of speB are in error. Within the limitations of the models used, we found no effect of cysteine protease on invasive streptococcal infection.

Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection.

Group A streptococcal (GAS) pharyngitis and the subsequent bacterial colonization of the human throat elicit an immune response that may precipitate acute rheumatic fever in a susceptible host. To study the bacterial determinants that influence throat colonization and induction of humoral immunity, we characterized the behavior of GAS strains in a baboon model. An M-type 3 clinical isolate of GAS typical of strains that cause pharyngitis and invasive infection was recovered from the pharynx of six out of six baboons for at least 6 weeks after oral inoculation. By contrast, an isogenic mutant deficient in M protein failed to colonize most animals or was rapidly cleared. An isogenic mutant deficient in hyaluronic acid capsule colonized five out of six animals, but only persisted in the pharynx for 14-21 days. Colonized animals developed serum antistreptolysin O (SLO) and anti-M protein immunoglobulin (Ig)G. The kinetics of the antibody responses were similar to those seen after human infection. Peak titres increased with the duration of throat carriage. Colonization with GAS prevented recurrent colonization after challenge with the homologous wild-type strain, but not after challenge with a strain of different M protein type. Early clearance of the M protein-deficient strain was associated with increased susceptibility of this strain to phagocytic killing in non-immune serum, whereas clearance of the acapsular strain was associated with increased susceptibility to phagocytic killing in the presence of specific antibody. These studies support critical and distinct effects of the GAS M protein and capsule on throat colonization and induction of humoral immunity in a model that reproduces important features of pharyngeal colonization and immune response following human infection.

Molecular analysis of the capsule gene region of group A Streptococcus: the hasAB genes are sufficient for capsule expression.

Enzymes directing the biosynthesis of the group A streptococcal hyaluronic acid capsule are encoded in the hasABC gene cluster. Inactivation of hasC, encoding UDP-glucose pyrophosphorylase in the heavily encapsulated group A streptococcal strain 87-282, had no effect on capsule production, indicating that hasC is not required for hyaluronic acid synthesis and that an alternative source of UDP-glucose is available for capsule production. Nucleotide sequence and deletion mutation analysis of the 5.5 kb of DNA upstream of hasA revealed that this region is not required for capsule expression. Many (10 of 23) group A streptococcal strains were found to contain insertion element IS1239' approximately 50 nucleotides upstream of the -35 site of the hasA promoter. The presence of IS1239' upstream of hasA did not prevent capsule expression. These results elucidate the molecular architecture of the group A streptococcal chromosomal region upstream of the has operon, indicate that hasABC are the sole components of the capsule gene cluster, and demonstrate that hasAB are sufficient to direct capsule synthesis in group A streptococci.

Molecular analysis of the role of the group A streptococcal cysteine protease, hyaluronic acid capsule, and M protein in a murine model of human invasive soft-tissue infection.

Human invasive soft-tissue infections caused by group A Streptococcus are associated with significant morbidity and mortality. To investigate the pathogenesis of these serious infections, we characterized the host response to bacterial challenge with an M-type 3 isolate recovered from a patient with necrotizing fasciitis, or with isogenic gene replacement mutants deficient in cysteine protease, hyaluronic acid capsule, or M protein in a murine model of human invasive soft-tissue infection. Animals challenged with the wild-type or cysteine protease-deficient strain developed spreading tissue necrosis at the site of inoculation, became bacteremic, and subsequently died. Histopathologic examination of the necrotic lesion revealed bacteria throughout inflamed subcutaneous tissue. Arterioles and venules in the subcutaneous layer were thrombosed and the overlying tissue was infarcted. In contrast, animals challenged with either an acapsular or M protein-deficient mutant developed a focal area of tissue swelling at the site of inoculation without necrosis or subsequent systemic disease. Histopathologic examination of the soft-tissue lesion demonstrated bacteria confined within a well-formed subcutaneous abscess. We conclude that the group A streptococcal hyaluronic acid capsule and M protein, but not the cysteine protease, are critical for the development of tissue necrosis, secondary bacteremia, and lethal infection in a murine model of human necrotizing fasciitis.

Structure of the has operon promoter and regulation of hyaluronic acid capsule expression in group A Streptococcus.

Group A streptococcal strains vary widely in the amount of hyaluronic acid capsule they produce, although the has operon, which encodes the enzymes required for hyaluronic acid synthesis, is highly conserved. The three genes making up the has operon are transcribed from a single promoter located upstream of the first gene in the operon, hasA. To investigate transcriptional regulation of capsule synthesis, we studied the structure and function of the has operon promoter sequences from two strains of group A Streptococcus: a highly encapsulated M-type 18 strain and a poorly encapsulated M-type 3 strain. Transcriptional fusions of the has operon promoter to a promoterless chloramphenicol acetyltransferase gene were constructed in a temperature-sensitive shuttle vector. The influence of promoter structure on has operon transcription was reflected by chloramphenicol acetyl transferase activity in cell lysates of Escherichia coli harbouring the recombinant plasmids and in group A Streptococcus after integration of the promoter fusions into the streptococcal chromosome. Fusions including as few as 12 nucleotides upstream from the -35 site of the has promoter exhibited full activity, indicating that sequences further upstream do not affect has gene transcription. A transcriptional fusion of the has promoter from the highly encapsulated M-type 18 strain was threefold more active than a similar construct from the poorly encapsulated M-type 3 strain. Analysis of the promoter sequences for the two strains revealed differences in three nucleotides in the -35, -10 spacer region of the promoter and in four nucleotides in the +2 to +8 positions relative to the start site of hasA transcription. To determine the relative importance of the two groups of nucleotide substitutions, chimeric promoter sequences were constructed in which either of the two clusters of variant nucleotides from the M18 has promoter was substituted for the corresponding positions in the M3 has promoter. Analysis of these chimeric promoter fusions showed that sequence changes in both regions influenced promoter strength. These results define the limits of cis-acting chromosomal sequences that influence transcription of the has operon and indicate that the fine structure of the promoter is an important determinant of capsule gene expression in group A Streptococcus.

Cloning, sequence analysis and expression of the group A streptococcal guaB gene encoding inosine monophosphate dehydrogenase.

Inosine monophosphate dehydrogenase (IMPDH) is an essential enzyme in the biosynthesis of purines. We cloned a group A streptococcal (GAS) DNA fragment containing an open reading frame similar to other bacterial guaB genes encoding IMPDH. The GAS guaB consists of 1479 nucleotides encoding a protein of 493 amino acids. Expression of the GAS guaB in an Escherichia coli guaB mutant restored IMPDH activity, confirming the function of the gene product and demonstrating that the GAS enzyme is active in a heterologous bacterial host. Restriction mapping and Southern hybridization analysis of GAS chromosomal DNA localized guaB to a site approximately 5 kb from the hasA and hasB genes which encode enzymes necessary for hyaluronic acid capsule synthesis.

Zollinger-Ellison syndrome in a patient with normal screening gastrin level.

A 59-year-old female presented with multifocal peptic ulcer disease and diarrhea. A fasting serum gastrin level obtained while the patient was receiving no antacid therapy was normal. A secretin stimulation test was positive. A small gastrinoma was found in the anterior duodenal wall at exploratory laparotomy. Normal fasting gastrin levels do occur in patients with overt Zollinger-Ellison syndrome and should not deter further investigation if clinical suspicion of this syndrome is high.