Search

Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complemented galE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-type Salmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuni encoded this epimerase. We constructed a C. jejuni galE mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galE gene in the synthesis of C. jejuni LPS. The galE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

Hamster trachea epithelial (HTE) cells were employed as a model system for Mycoplasma pneumoniae pathogenesis. To more closely mimic natural infection, M. pneumoniae was forced to rely upon host cells (as opposed to the growth medium) for nutrients, and infections were initiated with relatively low mycoplasma doses and monitored for extended time periods. A time- and dose-dependent decline in the viability of infected cells was observed; however, viability never declined below 50% of that in uninfected controls. Protein and RNA synthesis actually increased above control levels in infected cells, despite a concomitant decrease in viability. This response was pronounced at higher multiplicities of infection but was only transient at lower doses. In parallel studies in which a culture medium capable of supporting M. pneumoniae growth was used, loss of viability was accelerated. With a low-dose infection a transient increase followed by a precipitous decline in macromolecular synthesis was observed, relative to that in uninfected controls. At higher doses, however, macromolecular synthesis decreased dramatically and in proportion to the loss of viability. The requirement for HTE cells for mycoplasma growth under the experimental culture conditions was demonstrated by quantitating viable mycoplasmas in the culture medium in the presence or absence of HTE cells over 4 days. The increase in mycoplasma number was negligible in the absence of HTE cells, while a 30-fold increase was observed in the presence of HTE cells. These findings demonstrate the feasibility of long-term, low-dose studies of M. pneumoniae pathogenesis with trachea epithelial cells and a nonpermissive culture medium. This experimental system should facilitate the elucidation of the mechanism(s) responsible for host cell injury, and perhaps reveal how host cells respond to infection.

The susceptibility of HeLa cells to Mycoplasma pneumoniae-induced injury was examined. Infections were initiated with relatively low mycoplasma doses, carried out in a culture medium incapable of supporting M. pneumoniae replication in the absence of host cells, and monitored for up to 10 days. Under these conditions, a time- and dose-dependent decline in the number of viable host cells compared with that of uninfected controls was observed. The effect of M. pneumoniae infection on host cell macromolecular synthesis was also evaluated. At high doses of infection, synthesis of both protein and RNA declined rapidly relative to that in control cells. At lower doses there was a biphasic response in protein synthesis, which was substantially lower than that in the uninfected control by day 1 postinfection, returned to control levels by day 4 postinfection, and was again less than that in control cells by day 7 postinfection. In contrast, no transient recovery was observed in RNA synthesis, which declined very gradually over 7 days in infected HeLa cells compared with that in uninfected control cells. The ability of HeLa cells to support the proliferation of M. pneumoniae under these experimental conditions was demonstrated by quantitation of mycoplasma CFU in the nonpermissive medium in the presence or absence of HeLa cells. A negligible increase in the number of M. pneumoniae was observed over 4 days when HeLa cells were absent, while CFU increased by almost 20-fold when M. pneumoniae was cultured in the presence of HeLa cells. The susceptibility and response in macromolecular synthesis in M. pneumoniae-infected HeLa cells differed from that recently described for a nontransformed culture of hamster trachea epithelial cells under the same experimental conditions (Y.-Y. Chen and D.C. Krause, Infect. Immun. 56: 570-576, 1988), underscoring the importance of the choice of host cell for in vitro modeling of M. pneumoniae pathogenesis.