Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

In vitro assessment of cellular immunity in human coccidioidomycosis: relationship between dermal hypersensitivity, lymphocyte transformation, and lymphokine production by peripheral blood mononuclear cells from healthy adults.

Dermal hypersensitivity in coccidioidomycosis was compared with three simultaneous measures of in vitro cellular immunity using 35 healthy donors living in an area endemic for coccidioidomycosis. Twenty donors had greater than 5 mm induration to usual-strength spherulin and were considered skin test-positive. Mononuclear cells from these individuals were more responsive by lymphocyte transformation (12,541 +/- 3746 vs. -112 +/- 260 cpm, P = .007) and produced significantly more interleukin-2 (3481 +/- 1067 vs. -5 +/- 69 cpm, P less than .001) and interferon-gamma (1831 +/- 481 vs. 75 +/- 58 pg/ml, P less than .001) than cells from skin test-negative donors in response to a coccidioidal antigen. However, the correlation between the skin test size and the magnitude of the in vitro response among skin test-positive donors was poor (R2 = 0.08, P = .24). These results indicate that healthy individuals with dermal hypersensitivity to Coccidioides immitis can be distinguished from those without hypersensitivity by their cellular in vitro response to a coccidioidal antigen.

Intestinal parasites in Central American immigrants in the United States.

We describe the results of ova and parasite examinations of 216 Central Americans who were seen at a Los Angeles, Calif, clinic during an 8-month period. Among the 125 immigrants from Central America, intestinal parasitic prevalence was 53%. Pathogens were found in 45% and multiple pathogens in 21%. Of the 91 US born Central American children, parasite prevalence was 14%. Pathogens were found in 12%. The most common pathogens were Trichuris trichiura, Giardia lamblia, and Ascaris lumbricoides. Giardia lamblia was more prevalent in the younger than 5-year-old age group, and helminths were more prevalent in the 6- to 10-year-old age group. No helminths were found in immigrants who had been in the US for more than 3 years. Gastrointestinal symptoms did not correlate with prevalence of parasites. The high prevalence of intestinal parasites supports previous recommendations for screening; decisions for screening should be based on morbidity (probably low), efficacy and safety of treatment (good), and costs.

Surface attachment of a sediment isolate ofEnterobacter cloacae.

Enterobacter cloacae was recovered from surface sediments of a flood control channel in an area where freshwater runoff mixed with coastal seawater. Cells of this bacterium elaborated an extensive capsule when cultured under laboratory conditions designed to promote extracellular polysaccharide production. Colonization of glass surfaces by cells was similar under aerobic and anaerobic conditions. Temperature exerted little effect on maximum adherent cell density in the range of 15-25°C. The availability of organic nutrients also had little influence on the tendency of cells to adhere to surfaces. Maximum adherent cell densities decreased (76%) as salinity increased from 0 to 12‰ The results suggest that cells ofE. cloacae are suitably adapted to maintain a sessile existence in brackish water sediments of temperate coastal areas.