Publications

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract.

is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant strains due to its bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both and Using isogenic DAP-susceptible and DAP-resistant strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

Objectives

Among viridans group streptococcal infective endocarditis (IE), the Streptococcus mitis group is the most common aetiological organism. Treatment of IE caused by the S. mitis group is challenging due to the high frequency of β-lactam resistance, drug allergy and intolerability of mainstay antimicrobial agents such as vancomycin or gentamicin. Daptomycin has been suggested as an alternative therapeutic option in these scenarios based on its excellent susceptibility profile against S. mitis group strains . However, the propensity of many S. mitis group strains to rapidly evolve stable, high-level daptomycin resistance potentially limits this approach.

Methods

We evaluated the activity of 6 mg/kg/day daptomycin alone or in combination with gentamicin, ceftriaxone or ceftaroline against two daptomycin-susceptible S. mitis group strains over 96 h in a pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations.

Results

Daptomycin alone was not bactericidal and high-level daptomycin resistance evolved at 96 h in both organisms. Combinations of daptomycin + ceftriaxone and daptomycin + ceftaroline demonstrated enhanced killing activity compared with each antibiotic alone and prevented emergence of daptomycin resistance at 96 h. Use of gentamicin as an adjunctive agent neither improved the efficacy of daptomycin nor prevented the development of daptomycin resistance.

Conclusions

Addition of ceftriaxone or ceftaroline to daptomycin improves the bactericidal activity against S. mitis group strains and prevents daptomycin resistance emergence. Further investigation with combinations of daptomycin and β-lactams in a large number of strains is warranted to fully elucidate the clinical implications of such combinations for treatment of S. mitis group IE.

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of invasive diseases in neonates and severe infections in elderly individuals. GBS serine-rich repeat glycoprotein 1 (Srr1) acts as a critical virulence factor by facilitating GBS invasion into the central nervous system through interaction with the fibrinogen Aα chain. This study revealed that srr1 is highly conserved, with 86.7% of GBS clinical isolates expressing the protein. Vaccination of mice with different Srr1 truncated peptides revealed that only Srr1 truncates containing the latch domain protected against GBS meningitis. Furthermore, the latch peptide alone was immunogenic and elicited protective antibodies, which efficiently enhanced antibody-mediated opsonophagocytic killing of GBS by HL60 cells and provided heterogeneous protection against 4 different GBS serogroups. Taken together, these findings indicated that the latch domain of Srr1 may constitute an effective peptide vaccine candidate for GBS.

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.

Mast cell chymases and tryptases exhibit an intriguing but potentially confusing variety of forms and functions. Thanks to recent genetic and biochemical advances, a clearer picture of phylogenetic and functional relationships in this large group of mammalian enzymes is emerging. Furthermore, there is increasing appreciation of the diversity of these enzymes among human populations. In humans, there appears to be just one mast cell chymase but multiple expressed tryptases, some of which are allelic variants and others of which are products of separate gene loci. New biological tools, including the dipeptidyl peptidase I (DPPI)-null mouse in which the entire class of mast cell chymases appears to be functionally knocked out, are helping to clarify the importance and specific roles of these most abundant of secreted mast cell proteins.

In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term "pancreasin" because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and gamma-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.

Human chymase is a chymotryptic serine peptidase stored and secreted by mast cells. Compared with other chymotryptic enzymes, such as cathepsin G and chymotrypsin, it is much more slowly inhibited by serum serpins. Although chymase hydrolyzes several peptides and proteins in vitro, its target repertoire is limited compared with chymotrypsin because of selective interactions in an extended substrate-binding site. The best-known natural substrate, angiotensin I, is cleaved to generate vasoactive angiotensin II. Selectivity of angiotensin cleavage depends in major part on interactions involving substrate residues on the carboxyl-terminal (P1'-P2') side of the cleaved bond. To identify new targets based on interactions with residues on the aminoterminal (P4-P1) side of the site of hydrolysis, we profiled substrate preferences of recombinant human chymase using a combinatorial, fluorogenic peptide substrate library. Data base queries using the peptide (Arg-Glu-Thr-Tyr-X) generated from the most preferred amino acid at each subsite identify albumin as the sole, soluble, human extracellular protein containing this sequence. We validate the prediction that this site is chymase-susceptible by showing that chymase hydrolyzes albumin uniquely at the predicted location, with the resulting fragments remaining disulfide-linked. The site of hydrolysis is highly conserved in vertebrate albumins and is near predicted sites of metal cation binding, but nicking by chymase does not alter binding of Cu2+ or Zn2+. A synthetic peptidic inhibitor, diphenyl N alpha-benzoxycarbonyl-l-Arg-Glu-Thr-PheP-phosphonate, was designed from the preferred P4-P1 substrate sequence. This inhibitor is highly potent (IC50 3.8 nM) and 2,700- and 1,300-fold selective for chymase over cathepsin G and chymotrypsin, respectively. In summary, these findings reveal albumin to be a substrate for chymase and identify a potentially useful new chymase inhibitor.