Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Structural specificity of diamines covalently linked to peptidoglycan for cell growth of Veillonella alcalescens and Selenomonas ruminantium.

Putrescine and cadaverine are essential constituents of the peptidoglycan of Veillonella alcalescens, Veillonella parvula, and Selenomonas ruminantium and are necessary for the growth of these organisms (Y. Kamio and K. Nakamura, J. Bacteriol. 169:2881-2884, 1987, and Y. Kamio, H. P?s?, Y. Terawaki, and L. Paulin, J. Biol. Chem. 261:6585-6589, 1986). In this study, the structural specificity of the diamine requirement for normal cell growth of these bacteria was examined by using a series of diamines with a general structure of NH3+ X (CH2)n X NH3+. Diaminohexane (n = 6) which was incorporated into the peptidoglycan was as effective as putrescine (n = 4) and cadaverine (n = 5) for normal cell growth. However, diaminopropane (n = 3) and diaminoheptane (n = 7) were less effective for growth than diaminohexane, although they were incorporated into the peptidoglycan to the same extent.     

Correlation between Possession of a Respiration-Dependent Na Pump and Na Requirement for Growth of Marine Bacteria

The possession of a respiration-dependent primary sodium pump and the requirement of Na for growth were investigated in bacterial isolates from marine environments. The bacteria in which NADH oxidase specifically required Na for maximum activity were believed to possess a primary sodium pump. All bacteria that failed to grow without the addition of NaCl possessed a primary Na pump. All bacteria that had no primary Na pump grew without additional NaCl. The primary Na pump seems to be involved in the Na requirement of marine bacteria, and this can be regarded as a criterion for the definition of marine bacteria.     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA.

RepA protein, essential for the replication of plasmid Rts1, was purified, and its binding to mini-Rts1 subregions was examined by a DNase I protection assay. RepA protected the incI and incII iterons, a region immediately upstream of the repA promoter, and a 10-base-pair region located between the most external incII iteron and a GATC box. The protection was less efficient when preheated RepA was used.     

Cadaverine covalently linked to a peptidoglycan is an essential constituent of the peptidoglycan necessary for the normal growth in Selenomonas ruminantium

Cadaverine links covalently to the D-glutamic acid residue of the peptidoglycan in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium (Kamio, Y., Itoh, Y., and Terawaki, Y. (1981) J. Bacteriol. 146, 49-53). This report clarifies a physiological function of cadaverine in this organism by using DL-alpha-difluoromethyllysine, which had previously been shown to be a selective irreversible inhibitor of lysine decarboxylase of Mycoplasma dispar (P?s?, H., MaCann, P.P., Tanskanen, R., Bey, P., and Sjoerdsma, A. (1984) Biochem. Biophys. Res. Commun. 125, 205-210). DL-alpha-Difluoromethyllysine is now shown to be a potent and irreversible inhibitor of lysine decarboxylase of S. ruminantium in vitro; however, it did not inhibit the transfer of cadaverine to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan. DL-alpha-Difluoromethyllysine at 5 mM markedly inhibited the growth of the bacterium and caused rapid cell lysis. Immediately before the cell lysis, almost all cells became swollen, and such cells showed a loosened envelope structure when studied by electron microscopy. The peptidoglycan prepared from the DL-alpha-difluoromethyllysine-treated cells did not have covalently linked cadaverine. The growth inhibition by DL-alpha-difluoromethyllysine was completely reversed by adding cadaverine (1 mM) to the medium. Furthermore, the exogenous cadaverine was exclusively incorporated into the peptidoglycan in the presence of DL-alpha-difluoromethyllysine (5 mM), and a normal peptidoglycan was synthesized. The cell lysis and the formation of an abnormal cell structure were completely prevented by cadaverine added to the medium. We conclude that cadaverine covalently linked to the peptidoglycan in S. ruminantium is an essential constituent of the peptidoglycan and is required for cell surface integrity and the normal growth of S. ruminantium.     

Molecular analysis of HLA-B39 subtypes

Serological studies have suggested the presence of a new HLA-B39 subtype (B39.2) in the Japanese population. To identify the new HLA-B39 subtype and compare it with an other HLA-B39 subtype (B39.1), the genes encoding HLA-B39.1 (B ^* 39013) and B39.2 (B ^* 3902) have been cloned from Japanese. We have sequenced these genes and completed the sequence of HLA-B39.1 (B ^*39011 ) gene from a Caucasian that was partially sequenced. Comparison of the sequence data revealed that B ^* 3902 and B ^* 39013 differ by three nucleotide substitutions which result in a two amino acids change at residues 63 and 67, while one silent substitution at codon 312 is found between B ^* 39011 and B ^* 39013. These results suggest that B ^* 3902 has evolved from B ^* 39013 rather than B ^* 39011.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94051 (HLA-B^*39013), M94052 (HLA-B^*39011), and M94053 (HLA-B^*3902).