Recent advances in the life sciences have stimulated speculation about the major developments to be expected in microbial pathogenesis. This speculation has focused particularly on how the new concepts and powerful techniques available will be harnessed to speed up a research process that has great promise, not only for pure research but also for the development of innovative anti-infectious drugs and vaccines. Celebrating the 100th General Meeting of the American Society for Microbiology (ASM) is a wonderful opportunity not only to reflect on our discipline, but also to pay tribute to this society for its tremendous impact in promoting research in microbial pathogenesis, and to many of its members, American and non-American, for their contributions that have shaped this discipline.

What Did We Learn from the Recent Past?

From its starting point as a largely descriptive approach carried out in animal models, microbial pathogenesis moved quickly, merging in the late seventies with molecular genetics. It is not possible to cite all the pioneers; however, if one name was to emerge, no one, I believe, would argue against giving credit to Stanley Falkow (a recent president of the ASM) for his early vision of the fantastic potential that was at hand. He coined the concept of molecular Koch postulates [1] that all of us practiced, sometimes without even realizing it. The phenomenal diversity of variation that was created in an increasing number of pathogens, by generating avirulent mutants or transformants expressing one or several virulence genes, quickly led to two major new developments: we realized that virulence genes are not necessarily constitutively expressed, and that pathogenic bacteria respond to their environment and adapt their strategies according to the local conditions prevailing in the host.

Many pioneers can be acknowledged for this advancement; let me emphasize here the contribution of John Mekalanos in shaping this concept and developing innovative techniques such as in vivo expression technology (IVET) [2]. Soon thereafter, it became obvious that animal models of infection were not easily adapted to the screening of several thousand strains, mutants, and transformants. This forced the development of rational cell assay systems that made such screening an endeavor of human dimensions. The eighties were, therefore, a time for the hesitant integration of cell biology and microbial pathogenesis. Shamelessly, we opened cell biology textbooks, often assisted by toxin specialists who had been flirting with eukaryotes for years, and, with childish pleasure, began mixing our nasty microorganisms with those splendid eukaryotes. What had begun as the design of easy screening assays quickly became a research topic per se. Invasive pathogens that have, by necessity, developed a complex - not to say complete - program of interactions with eukaryotic cells naturally became major tools to decipher the nature of these interactions [3]. Why should we not study in detail how bacterial virulence effectors interact with eukaryotic receptors, both from outside and inside those cells? "Cross-talk" became the word and cellular microbiology was born [4]. What's next?

Thanks to its potential for renewal and its transdisciplinary essence, which integrates molecular genetics, cell biology, structural biochemistry, immunology, and experimental medicine, microbial pathogenesis in its bright new clothes has become the figurehead of microbiology. This discipline continues to be attractive to young students and scientists, and has served as an incentive to create new research facilities and launch new programs and journals. As in anything else, openness and diversity are the keys to continued success; however, the field now needs to manage its rapid progress efficiently. One does not need a crystal ball to foresee the two major challenges of the new millennium.

Handling Excess Information: How Do We Do Genomics?

"Qui trop embrasse mal étreint" ("grab all lose all") - ancient wisdom entreats us to focus. The sequencing and annotation of genomes of the major pathogens and use of high-throughput techniques has catapulted us into a schizophrenic way of thinking. On the one hand we are fascinated by the generation of a fantastic amount of information concerning prokaryotes (i.e., genes and gene products potentially involved in pathogenesis) and their eukaryotic partners (i.e., cellular genes specifically expressed upon infection, detected in microarrays, and cellular "prey" picked up by bacterial "bait" in the two-hybrid system). Yet on the other hand, we tend to panic when the time comes to handle so much information and organize it into coherent areas of research aimed at further deciphering pathogenesis schemes. Will we be able to reconcile these apparently divergent paths? Yes, of course we will, because we have already practiced a large part of this postgenomic approach in the pregenomic period! The tools are available; it is simply a matter of better and more systematic organization, in an area in which bioinformatics has already proved invaluable. With pathogenicity islands [5], for example, some logic has already emerged in genome organization with regard to pathogenesis. Our increasing knowledge of the eukaryotic components involved in signaling, motility, trafficking, and maturation of compartments, life cycle and death pathways, control of gene expression and developmental pathways, and many other processes increases the likelihood of identifying the relevant target of pathogenicity factors, provided that feasible screening assays are developed to accomplish this endeavor. Subcellular microbiology is on its way.

Excessive complexity of systems

There are four main challenges connected with the "excessive complexity" issue:

(1) Deciphering the detailed molecular interactions between a pathogen's effectors (e.g., adhesins and invasins) and its cellular targets. Structural chemistry will soon become an integral part of our research strategies, as is already the case in studies of toxins.

(2) Integrating cellular microbiology into the broader scheme of host infection; in other words, how do we move from cellular microbiology to integrative tissue microbiology? Harry Smith must be credited for having constantly stimulated the community to look in vivo. Great progress has been made in detecting gene expression in cells and tissues, but one of the greatest achievements has certainly been the development of the negative screening technique known as signature-tagged mutagenesis (STM), which allows one to detect virulence genes in their relevant environment [6]. In addition, complex hetero-cellular assay systems are being developed to mimic tissue structures such as the barriers whose subversion is a key step to successful infection (i.e., in vitro modeling of the intestinal barrier or the blood-brain barrier), or mimic the cellular interactions that result in the development of an innate and/or adaptive immune response. This experimental movement from in vitro to in vivo is complemented by an opposite movement in which animal models of infection are being refined by combining microsurgery, implantation of microprobes coupled with devices for real-time titration of mediators, new powerful imaging techniques such as NMR/PET scans, a- and b- imaging, and molecular histopathology. In other words, introducing a clinical approach to the analysis of the development of infection.

(3) Addressing the concepts of host sensitivity and resistance to infections, and the immunoreactivity to pathogens, which can influence the severity of an infectious disease. Just as creativity has been required to elucidate the mechanisms of action of pathogenic bacterial effectors, similar efforts will be required for the development of ad hoc cell assay systems and animal models necessary to determine the function of an increasing number of candidate eukaryotic host genes that modulate the host's sensitivity to infectious diseases and dictate the host and organ specificity of the infectious agent. Transgenic and genetically altered animals will increasingly be part of the arsenal required to address these issues rigorously. Infection of model multicellular organisms such as Caenorhabditis elegans and Drosophila melanogaster, which are prone to "easy" genetic analysis, are also in the pipeline.

(4) Identifying factors required for the survival of pathogens in the environment ("vironmence" factors versus "virulence" factors) and evaluating the extent of overlap between these two gene pools. This will not be properly addressed unless we are able to develop innovative assays mimicking specific environmental conditions.

Conclusion

Discoveries cannot be foreseen and science cannot be programmed. However, trends can be identified that delineate the future challenges to be met, and I have tried to offer some suggestions here. Other viewpoints are also of great value because, again, diversity is a key issue for success. A forum sponsored by the ASM on microbial diversity [7] emphasized the important role of bacteria in the foundation of the biosphere. Let us not forget that, if we have learned a lot about bacterial pathogens, we know virtually nothing about the physiology of our own resident flora, and hope that research on this "good" flora will soon also benefit from the outstanding progress stimulated by the urgent need to control and eradicate the "bad" flora.