Role of ADP-ribosyltransferase activity of pertussis toxin in toxin-adhesin redundancy with filamentous hemagglutinin during Bordetella pertussis infection.

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two major virulence factors of Bordetella pertussis. FHA is the main adhesin, whereas PT is a toxin with an A-B structure, in which the A protomer expresses ADP-ribosyltransferase activity and the B moiety is responsible for binding to the target cells. Here, we show redundancy of FHA and PT during infection. Whereas PT-deficient and FHA-deficient mutants colonized the mouse respiratory tract nearly as efficiently as did the isogenic parent strain, a mutant deficient for both factors colonized substantially less well. This was not due to redundant functions of PT and FHA as adhesins, since in vitro studies of epithelial cells and macrophages indicated that FHA, but not PT, acts as an adhesin. An FHA-deficient B. pertussis strain producing enzymatically inactive PT colonized as poorly as did the FHA-deficient, PT-deficient strain, indicating that the ADP-ribosyltransferase activity of PT is required for redundancy with FHA. Only strains producing active PT induced a local transient release of tumor necrosis factor alpha (TNF-alpha), suggesting that the pharmacological effects of PT are the basis of the redundancy with FHA, through the release of TNF-alpha. This may lead to damage of the pulmonary epithelium, allowing the bacteria to colonize even in the absence of FHA.

The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination.

Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.

Term newborns who are at risk for sepsis: are lumbar punctures necessary?

OBJECTIVES: To determine: (1) whether a lumbar puncture (LP) is indicated in asymptomatic full-term newborns delivered by mothers at risk of intrapartum sepsis; and (2) whether gentamicin improves bacterial coverage for such newborns when used with ampicillin. DESIGN: A retrospective chart review from 1987 through 1993 of all newborns with positive blood and/or cerebrospinal fluid cultures in the first 7 days of life. METHODS: Pregnant women were screened in the second trimester for group B streptococci and given ampicillin during labor if two or more risk factors were present: group B streptococci colonization, maternal fever or leukocytosis, rupture of membranes at more than 18 hours, foul-smelling amniotic fluid, and fetal tachycardia. After sepsis evaluation (LP, blood culture, white blood cell count, and differential), asymptomatic infants received ampicillin and gentamicin for 48 to 72 hours unless cultures grew pathogens. RESULTS: Of approximately 24 452 full-term births in 7 years, 7% (1712) had evaluations for symptoms of sepsis, and 14% (3423) were asymptomatic but had evaluations for maternal risk factors. There were 11 cases of meningitis, all involving symptomatic newborns; 10 of these 11 had positive blood cultures for the same organism. In asymptomatic infants, none of the 3423 had meningitis (95% confidence interval, 0 to 0.0008), although 35 grew contaminants. Of 73 pathogens isolated from blood or cerebrospinal fluid, 7 (9.5%) were resistant to ampicillin. Addition of gentamicin provided coverage for only 2 of these 7 pathogens. Of 5135 infants who received ampicillin and gentamicin, only 2 required gentamicin for improved coverage. CONCLUSIONS: (1) LP is unnecessary in asymptomatic full-term newborns. (2) Empiric coverage for asymptomatic newborns with maternal risk factors need not include gentamicin at all hospitals, because it only improved the coverage of ampicillin alone from 90% to 93% of pathogens, but it exposed more than 5000 infants to the side effects of gentamicin. (3) The presence of leukopenia (<5000 white blood cells/mm) is highly predictive of bacteremia.

[A case of primary pulmonary hypertension in a 52-year old man]. / Przypadek pierwotenego nadcisnienia plucnego u 52-letniego mezczyzny.

The presented case of primary pulmonary hypertension is worth attention in view of rare incidence in men, especially those over 50 years of age. It was possible to observe the course of the disease and to perform examinations confirming the diagnosis. The unavoidable and rapid progression of lesions, extremely poor prognosis and lack of possibilities of causal treatment should be stressed.

[A case of Hashimoto struma coinciding with limited scleroderma]. / Przypadek wspólwystepowania wola Hashimoto i twardziny ograniczonej.

In a female patient aged 43 years severe hypothyroidism was caused by Hashimoto struma. She had also cutaneous changes suggestive of collagen disease. On the basis of clinical manifestations, laboratory investigations and histological examinations coexistence was diagnosed of two autoimmunological diseases. Stress is laid on diagnostic difficulties and choice of management.