Bernard Dixon

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Changing Pictures of Pathogenicity and Virulence

New techniques and perspectives are aiding progress and finding answers, but raising new questions as well

Bernard Dixon

A dialysis bag is an odd but appropriate artifact to symbolize the interface between two investigational techniques. Described during a recent meeting at the Royal Society in London, this one sits at the boundary between the chemical approach to microbial pathogenicity and the analysis of virulence through molecular genetics. While the former is already a mature discipline, the latter is in its initial, vigorous phase of growth.

The devisers of the dialysis bag are Carlos Orihuela and colleagues at the University of Texas Medical Branch, Galveston, and Information Dynamics, Inc., in Nassau Bay, Tex. They have been using it to explore the difference between the behavior of Streptococcus pneumoniae growing in vitro and in vivo. Inoculated with pneumococci, the bag is surgically implanted in the abdominal cavity of a mouse. Several hours later, it is recovered and the bacterial products evaluated.

In the study presented in London, the Texas group showed by SDS-PAGE and other techniques that production of several different proteins was enhanced significantly in vivo. The organisms also adhered 10 times more avidly to lung epithelial cells than those cultured in vitro. An analysis of virulence gene expression by Northern dot blotting revealed that transcription of the genes for pneumolysin and for synthesis of type 3 capsule more than doubled.

No doubt Harry Smith would have relished the opportunity to harness techniques of this sort when he pioneered the chemical study of pathogens some 40 years ago. The man who discovered why Brucella abortus preferentially attacks the fetal membranes of cattle, sheep, and goats (because they are rich in erythritol), Harry is now an emeritus professor at Birmingham University, Birmingham, United Kingdom. His continuing fascination with the specific determinants of microbial disease is attested by his role as co-organizer of the Royal Society meeting.

Our comprehension of microbial disease is, of course, now being revolutionized by the sequencing of the genomes of successive pathogens. But this is by no means the only development that is transforming the picture. Equally important is the emergence of powerful methods for identifying virulence genes and their expression in vivo. Thirdly, knowledge is burgeoning regarding the signals associated with the adhesion of bacteria to target cells, invasion, and interference with host cell functions.

While a combination of bench and animal studies led to the identification of nutritional factors such as erythritol in contagious abortion in cattle, other diseases have proved considerably more difficult to fathom. This is because many crucial aspects of communicable disease are not manifest in organisms cultured in laboratory glassware--even those freshly isolated from diseased tissues.

Screening with tissue-cultured host cells is superior in this sense to doing so with lifeless culture media. Nevertheless, it fails to reveal all virulence genes because such cells reflect neither the sophisticated architecture of the target organ nor the complex interactions between pathogens and host immune defenses.

The new techniques, however, allow experimenters to detect hitherto unrecognized pathogen genes and their expression during the course of an infection. They can, for example, go way beyond our knowledge of constitutive gene products to reveal genes which are expressed in vivo in an organ- or cell-specific fashion.

As Andrew Camilli of Tufts University, Boston, Mass., explained, in vivo expression technology (IVET) has been developed with a variety of reporter systems permitting either in vivo selection or ex vivo screening. ``Selectable gene fusion systems generally allow for the complementation of a bacterial metabolic defect that is lethal in vivo, or for antibiotic resistance during the course of in vivo antibiotic challenge.

``In contrast, the screenable gene fusion system uses a site-specific DNA recombinase which, when expressed in vivo, excises a selectable gene cassette from the bacterial chromosome. Loss of this cassette can then be either screened or selected for ex vivo.''

Investigators are using the recombinase-based IVET system (RIVET) to detect genes that are transcriptionally induced during an infection. It is sufficiently powerful to reveal those which are expressed at low levels and those which function only transiently. RIVET helps to demonstrate the spatial and temporal pattern of gene expression as an infection progresses.

Signature-tagged mutagenesis (STM), another novel methodology, allows investigators to test many different insertional mutants simultaneously in a living host, which selects against strains whose virulence has been attenuated. It was initially validated using salmonella and a mouse model of typhoid fever by the isolation of many genes now known to be important for virulence (see p. 15).

Brian Sheehan and his colleagues have applied STM to study the highly contagious, rapidly fatal pleuropneumonia caused by Actinobacillus pleuropneumoniae in pigs. Working at the Imperial College School of Medicine in London and the Royal Veterinary College, Hatfield, U.K., they first screened 48 sequence-tagged mini-Tn10 mutants by intratracheal inoculation.

This led them to identify four attenuated mutants and to the DNA flanking each transposon insertion site. In one, the transposon had inserted in a gene not resembling any to be found in the public databases. The others contained insertions in loci encoding an ABC-type transporter, an NAD(P) transhydrogenase, and TonB, which had previously been implicated in the virulence of several different organisms.

Sheehan and his coworkers pointed out that S. typhimurium TonB mutants were attenuated when introduced by the mucosal route but not when given intraperitoneally. ``TonB was not identified during the STM analysis of S. typhimurium, emphasising the importance of the route of infection in identifying genuine modulators of bacterial virulence,'' they concluded. ``STM holds promise for identifying A. pleuropneumoniae genes for natural porcine infection, which may be exploited in designing strategies to prevent the disease.''

STM is clarifying the relationships between salmonellae and their target species, too. Tim Wallis and colleagues at the Institute of Animal Health, Compton, U.K., have used it to identify two S. dublin mutants with apparently altered host-specific phenotypes.

One, which seemed to be attenuated only in mice, contained an insertion in the sseD gene in Salmonella pathogenicity island 2. This locus (SPI-2) was previously associated with the systemic survival of S. typhimurium in mice. The other mutant, attenuated only in calves, had the insertion upstream of the Vibrio cholerae gene hcp, which had not previously been identified in salmonellae.

Wallis and collaborators believe that both genes code for secreted proteins. In contrast to their initial suspicions, they discovered that the sseD mutant was avirulent in both mice and calves. They believe their work provides the first evidence of the importance of SPI-2 as regards systemic salmonellosis in calves.

The Compton team also used in vivo tests to confirm that the putative hcp mutant was attenuated in calves but remained virulent in mice. This, in turn, is the first piece of work showing that the hcp locus influences virulence and, potentially, serotype-host specificity.

Techniques and advances of this sort are indeed impressive. Yet as Richard Moxon of Oxford University pointed out during the Royal Society meeting, they do not solve the entire problem of how microbes cause disease. Practical and semantic difficulties remain in distinguishing so-called virulence factors from the many determinants promoting general fitness. ``Despite the awesome power of molecular biology,'' Mozon said, ``we are still struggling to come to grips with the simple question of `what is a pathogen?'''

As in many other sectors of biology, we need to remain vigilant over the seductions of reductionism and determinism.