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Of Microbes and Men. War and Peace on the Mucosal Surfaces

Philippe Sansonetti

Of Microbes and Men: War and Peace on the Mucosal Surfaces

Inaugural lecture delivered on Thursday 20 November 2008

Philippe Sansonetti
Traduction de Liz Libbrecht

Texte intégral

1Mr Administrator,
Dear colleagues,
Ladies and gentlemen.

2Thank you Mr Administrator and dear Pierre for this laudatory and warm introduction. Thank you, dear colleagues, for deeming me worthy of being a member of the Collège. I am sincerely grateful to those of you who have directly guided and supported me in the project of creating a Chair of Microbiology and Infectious Diseases.

Peace and its compromises, the primate-microbe hybrid Human

3Humans evolve in a microbial world: bacteria, viruses, bacterial viruses (bacteriophages), yeasts and fungi. To that I can add unicellular and multi-cellular parasites, even though they do not strictly speaking fit the definition of microbes as they are a major cause of infection. In today's lecture I will talk a lot about bacteria, as that is what most of my scientific work has focused on.

4As soon as living beings became multi-cellular, they had to socialise with microbes, the planet’s first inhabitants, and establish with them a state of commensalism, even symbiosis. Model multi-cellular beings such as the Caenorhabditis worm and the Drosophila fly, have a commensal microbiota – that is the term now used to define the resident microbial flora – and are sensitive to pathogens. The systems governing the management of this interface, born of the adaptation of some of the most fundamental mechanisms of development, have been preserved remarkably well throughout evolution, from insects to higher primates. “Nothing in biology makes sense except in the light of evolution”, said Theodosius Dobzhansky. The human-microbe co-evolution did not stop at the recognition and eradication of pathogens; it has also led to tolerance of commensal microbiota. Our organism’s microbiological monitoring system is constant. Si vis pacem, para bellum. That is the paradox around which our immune system has developed: “overcome ignorance, learn tolerance, adjust the response to the gravity of the offence”, that is a societal challenge that microbes raise.

5Alongside acute infectious diseases, chronic colonizations exist that give rise to significant complications in a limited number of cases only. Thus, in the poor regions of the world, the Helicobacter pylori bacteria, a gastric pathogen, can colonize the population from a very young age, but will only cause complications (peptic ulcer, stomach cancer) in a small proportion of people. Should it be described as a commensal bacteria transgressing the boundary, or as a furtive pathogen ensuring its prolonged survival? Should we see individuals’ varying sensitivity in relation to their genetic baggage, or to environmental factors? Probably all four at the same time – which underscores the need for multi-disciplinary research in the field of infectious diseases.

  • 1 Human papillomavirus. This virus is responsible for a very large number of sexually transmitted dis (...)

6Note here that close to 20% of cancers are of infectious origin, and can therefore effectively be prevented when a vaccine is available. Hepatitis B and HPV1 vaccine sceptics would do well to ponder this fact.

7To complicate the picture, certain pathologies like chronic inflammatory bowel diseases (Crohn’s disease for example) reflect poor management of the interface with our commensal microbiota. The genetic analysis of these diseases is starting to provide us with candidate genes, all providing leads pointing towards the molecular and cellular nature of this interface. Are we going to have to reconsider our concepts and definitions of infectious diseases? Genetic sensitivity to usually non-pathogenic microorganisms in the environment, opportunistic infections of immune-depressed subjects, polymicrobial infections: exceptions to Koch’s postulate abound. The paths of the host-microbe co-evolution often remain unfathomable, and only an evolutionist perspective rooted in mutual selective pressure makes sense.

  • 2 François Jacob, Le Jeu des possibles. Essai sur la diversité du vivant, Paris, Fayard, 1981.

8“It is not the strongest of the species that survives, nor the most intelligent… It is the one that is the most adaptable to change”, wrote Charles Darwin. “Evolution proceeds like a bricoleur who, over millions and millions of years, slowly alters his work, constantly making adjustments, cutting this bit off, lengthening that one, finding every opportunity to adjust, transform, and create”, François Jacob wrote in Le Jeu des possibles2. Without this interpretation, it is impossible to understand how our interface with microbes developed: for example, how the path along which the fly’s dorsoventral developed can have come to guide the perception of microorganisms even in mammals; or how a bacterial flagellum can have transmuted into a toxin secretion system, or vice versa. The host-microbe interface is everything like the bricoleur and nothing like Intelligent Design. As Charles Darwin also said, “nature is prodigal in variety, though niggard in innovation”.

9Basically, human beings are primate-microbe hybrids. We host ten times more bacteria than we have somatic and germinal cells. The intestinal tract hosts most of this microbiota, reaching the astronomical figure of 1014 bacteria (one hundred thousand billion). Extrapolating molecular identification results suggests that we host between 15,000 and 30,000 different species. One can therefore consider that the human genome sequence will not be complete as long as we don’t have that of its microbiome, that is, a gene pool probably 100 times greater than the human genome. This is a real challenge, as the microbiota varies from one individual to the next, from one region to the next, and from one anatomic site to the next.

10These terrae ingognitae, new frontiers of microbiological research, are already the subject of metagenomic research by several North American, European, Japanese and Chinese consortiums. My first lecture on microbial commensalism will go hand in hand with a seminar held by our colleague from INRA, Joël Doré, on the expectations formed around these metagenomic analyses as well as their limits.

11Let me simplify this... The intestinal microbiota comprises two main groups: firmicutes, anaerobic gram-positive bacteria, and bacteroidetes, anaerobic gram-negative bacteria. The proteobacteria we are most familiar with, such as Escherichia coli, are actually an ultraminority – at least in homeostasis, for in certain pathological situations like inflammatory bowel diseases, balances are disrupted and bacteroidetes make way to proteobacteria. Who is the egg, who is the hen? Does the inflammatory disease cause the microbiota imbalance, or does the imbalance cause the inflammatory disease?

12Did I say “simplify…”? Let’s try. The intestinal microbiota has a similar global metabolic activity to an organ like the liver. This supernumerary organ has many functions. It maintains a barrier effect against allogeneic, potentially pathogenic microorganisms. It ensures the homeostasis of the intestinal epithelial barrier by stimulating its restitution in cases of alteration, but also takes care of the development and maintenance of the mucosal immune system and of the sub-epithelial vascular network. It plays a key role in nutrition and the metabolism: hydrolysis and fermentation of complex polyosaccharides (especially plant sugars for which our enzymatic equipment is inadequate), vitamin biosynthesis, production of light-chain fatty acids that represent a major source of nutrition for the epithelium, detoxification of food xenobiotics. This is therefore a case no longer of commensalism in the generic sense of the term, but of real symbiosis.

13What would we be without our microbiota! Yet perhaps it is becoming an impediment in a Western world where hygiene and dietary imbalances are prevalent. Could this supernumerary organ make us obese or diabetic, as recent work by Jeff Gordon's group in the USA suggests?

14We recently embarked on a molecular and cellular analysis of the mechanisms and signals through which the microbiota regulates the homeostasis of the intestinal crypt-villus axis, from the crypt’s stem cells to the differentiated epithelial cells at the surface. We are also seeking to understand how pathogenic microbes disrupt this homeostasis. The opening of this developmental microbiology-biology interface illustrates the wealth of research opportunities afforded by microbiology.

The war and its confrontations, a genetic programme for virulence

15Let us now look at pathogens. Although they constitute a minority in this microbial environment, they have a major impact: every year, infectious and parasitic diseases kill approximately 15 million people, including 12 million children, not to mention the morbidity and devastating sequela left by some of them. The economic and social cost of this disaster is enormous, particularly in countries of the South where more than 90% of infections occur, curbing the development of the poorest countries. It is better to know these pathogens if we are to fight them. Can we actually draw a line between good bacteria – the microbiota – on the one hand, and bad ones – those that are pathogenic – on the other?

  • 3 Charles Nicolle, Destin des maladies infectieuses, Paris, Félix Alcan, 1933.

16Charles Nicolle was already asking this question in Destin des maladies infectieuses3 (The Destiny of Infectious Diseases), a collection of lectures he gave at the Collège de France in the early 1930s. Within the complexity of the microbial world, from the criteria of form, of metabolic pathways and antigenic specificities – what he called “the mosaic of powers” – only one stood out in his reflection. “What makes the specificity of a pathogenic agent”, he said, “are its virulent properties”. In this respect he echoed Émile Roux: “A microbe’s virulence is its ability to live in the organism of superior beings and to secrete poison therein”. Little by little, Louis Pasteur’s microbial theory of infectious diseases took shape thanks to the work and reflection of his students and successors, including Élie Metchnikov, who 100 years ago was awarded a Nobel Prize for the discovery of phagocytosis; thanks also to the work of Robert Koch and his school, from Paul Ehrlich to Emil von Behring, the latter having demonstrated that the seroneutralization of the tetanus or diphtheria toxin was enough to inhibit the disease, providing the brilliant proof that a factor of virulence constituted the molecular signature of a pathogen. And Charles Nicolle who topped it off with formidable intuition: “There are therefore good reasons to think that virulence is linked to a physical medium. Do we not sometimes see it undergo sudden variations, to which the meaning and name of “mutations” can legitimately be given, and do these sudden variations, in addition to the adaptation to a new being, then not translate into the acquisition of new pathogenic properties in relation to the animal species that it [the pathogenic bacteria] ordinarily infects. 1932, and it’s all there! ... Except for the physical medium.

17In London, almost concurrently, Frederick Griffith discovered this transformation in the pneumococcus, allowing three researchers at Rockefeller University in New York (Oswald Avery, Colin McLeod and Maclyn McCarty) to demonstrate that Griffith’s transforming principle – capable of restituting the expression of its capsule, and therefore its virulence, to an acapsulated pneumococcus strain – was destroyed by the DNase enzyme. This experiment proved that the physical medium of virulence was deoxyribonucleic acid, which “accessorily” became THE heredity medium!

18It was soon thereafter discovered that the key factors of virulence, like the diphtheria toxin, are coded by mobile genetic elements like the bacteriophage beta. Then, in the 1950s, came the molecular genetics revolution: André Lwoff, Jacques Monod, François Jacob, Elie Wolmann and many others developed the concepts and tools with which could be linked, if not Escherichia coli and the elephant, then at least Escherichia coli and pathogenic micro-organisms.

19Stanley Flakow then had the intuition that this emerging molecular genetics would allow for microbes’ pathogenic power to be deciphered. The last chapter of his 1975 book Infectious Multiple Drug Resistance is dedicated to the beginnings of this genetic study, following an exhaustive review of the work on the genetics of bacteria’s resistance to antibiotics, which was the emerging question of the 1970s. This sounded the beginning of the end of the wave of optimism that had too hastily predicted the end of infectious diseases. And that is how, as a young intern at the Hôpitaux de Paris, I decided to take classes at the Pasteur Institute to learn, thanks in particular to Agnès Ullmann, Aline Brachet and Yves Chabbert, the necessary basics to begin this analysis. I will revert to this.

20What did molecular genetics, followed by pathogen genomics, teach us? Escherichia coli proved to offer an excellent model from the start. This single species includes both commensal isolates and pathogenic isolates; and the latter can be broken down into several intestinal, urinary and septicemic pathovars responsible for meningitis. It soon became apparent that beyond the metabolic and antigenic properties often found to be faulty – which Charles Nicolle would not have denied – each of these pathovars has a complex genetic signature corresponding to the genomic accretion of elements: some are mobile, the bacteriophages and plasmids; others are fixed in the chromosome but probably of phage origin, the “pathogenicity islands”. This pathogenecity developed through quantum leaps, culminating in the “final touch” with which this whole system was put under the control of a strict regulatory hierarchy responding to environmental conditions (Figure 1). Sequencings and pan-genomic analyses have perfectly confirmed this concept which reflects the existence of constant gene flows. One of my lectures will deal with the genetic and molecular identity of pathogens.

Figure 1. Mechanisms involved in the evolution of bacterial genomes

Figure 1. Mechanisms involved in the evolution of bacterial genomes

21Of course, some questions remain:

  • What is the origin of these foreign genes?

  • Does this model apply to all pathogens? The simple answer is “often but not always”. Model organisms are ideal research objects provided one is able to move away from them.

    • 4 The virus responsible for bird flu.

    Finally, did viruses undergo a similar genomic evolution? Yes, most probably, in the case of large DNA viruses like Pox and Herpes, which acquired genes from their cellular partners, essentially serving them to deceive our immune system. The answer is not so clear for RNA viruses, where intra-specie recombinations and the fickleness of RNA polymerase contribute to generating the necessary diversity for adaptation to hosts and species jumps. What is missing in the H5N14 virus’s “specifications” to cause a flu pandemic? We will endeavour to answer to this question.

  • 5 Tolerogenic: which creates a state of immune tolerance to an antigen or to a microorganism.

22Let us return to the heart of the paradox of commensalism and pathogenicity, which are managed by the same immunological surveillance systems. Intestinal commensals establish a subtle molecular dialogue with the epithelium through the surface mucus filter, on which they are organized into complex biofilms. They are kept at a respectable distance by a gradient of antimicrobial epithelial factors, and their proximity and density are sensed through the constant sampling of molecules peculiar to the prokaryotic world. Through the analysis and epithelial filtering of these signals, this molecular dialogue results in a tolerogenic situation5 for the microbiota. Most likely very similar mechanisms govern the tolerance of other microbiotas, particularly the cutaneous microbiota.

23As for pathogens, they have an array of virulence factors allowing them to access the host’s surfaces, to adhere to them, potentially to invade them and, using dedicated systems, to inject toxins that will destroy the epithelial barrier and its immune defence systems. Together these effects are perceived as a danger signal by the host, as a signal that an alert threshold has been breached: hence an inflammatory microbicidal innate immune response (Figure 2), yet one that is also destructive, and largely responsible for the symptoms and lesions of the disease. It is time to discuss the work of my group on Shigella.

  • 6 PAMPs: pathogen-associated molecular patterns, molecules belonging to the prokaryotic world (lipopo (...)

Figure 2a. Commensalism6

Figure 2a. Commensalism6

Commensals: Absence (limitation) of virulence factors. Less agonist PAMPs (not constant). Sequestration, less TLR activity. Life in biofilms on the surface of the mucus. Controlled dissemination and sampling of the PAMPs and prokaryotic signalling molecules.

  • 7 NLR : nod-like receptors, cytoplasmic sensors.

Figure 2b. Pathogenicity7.

Figure 2b. Pathogenicity7.

Pathogens: Mucinases. Adhesins. Invasins. Type III/IV secretion systems. Hemolysins Massive receptor engagement by microbial products.

Shigella, model for deciphering the virulent identity of a microorganism

24In 1978, I contacted Léon LeMinor who was head of the Enterobacteriaceae Unit at the Pasteur Institute and told him about my wish to apply my fresh knowledge in genetics to the study of a pathogen. I chose Shigella, the bacteria responsible for bacillary dysentery, an acute inflammatory colitis characterised by abscesses and ulcers reflecting the bacteria’s capacity to break through the intestinal epithelium and invade cells. The presence of a bacillus coupled with a massive efflux of polynuclear neutrophils in patients’ stools had been described by Chantemess and Widal, but the Japanese microbiologist Shiga was the one who identified Shigella dysenteriae, the bacillus named after him. Shigellosis is an endemic disease caused by Shigella flexneri and Shigella sonnei, which still kills hundreds of thousands of infants and children every year and is the primary cause of malnutrition in the most deprived regions of the world. Epidemic, multi-resistant, caused by Shigella dysenteriae 1 or the Shiga Bacillus, like cholera it decimates refugee camps with very high attack and mortality rates. Scientifically speaking, Shigella also had a double advantage: very close to Escherichia coli, it could be genetically manipulated like this model microorganism. Invasive, capable of penetrating the epithelium of the human colon, it allowed for a large number of possible stages of a microorganism’s virulence to be summarized. This proved to be the right choice.

  • 8 Plasmid: genetic element independent from the bacterial chromosome, capable of replicating autonomo (...)
  • 9 Apoptosis: programmed cellular death which, unlike necrosis, involves not cell lysis but its fragme (...)

25I quickly showed that the genetic support of the Shigella invasion was extra-chromosomal: a large 220 kb plasmid8 allowed the bacteria to enter the host’s epithelial cells. At the time, this provided the first evidence that invasiveness could be genetically analyzed. Bolstered by this discovery, I went on to do my post-doctoral placement in Samuel Formal’s group in Washington. Within two years, I confirmed this plasmid’s role. I transferred it to the Escherichia coli using assisted conjugation and – thanks to an interrupted conjugation approach using Hfr donor Shigella strains in this same Escherichia coli – I mapped most of the chromosomal loci, reconstructed a completely virulent Shigella strain from scratch and thereby identified, other than the virulence plasmid, all the loci defining the genetic identity of Shigella as a pathogen: a sort of genomics before its time. With these data thus began the “Shigella saga” upon my return to the Pasteur Institute. Hélène d’Hauteville, Joëlle Mounier, Philippe Clerc, Tony Maurelli, Lina Bernardini, and I described the basic elements of cellular invasion: cloning of the plasmid sequences needed for the bacteria to enter cells through a macropinocytosis process relying on cellular actin (Figure 3a), discovery of the intracytoplasmic motility and of the bacteria’s movement from one cell to the next, completely new phenotype also linked to the mobilization of the cellular actin cytoskeleton (Figure 3b). Arturo Zychlinsky and I showed for the first time that a bacterial pathogen programmed the apoptotic death9 of the macrophages it infected. Paradoxically, this process went hand in hand with the massive liberation of pro-inflammatory IL-1 beta cytokine due to the activation of the Caspase 1 enzyme. This pro-inflammatory apoptosis was considered both contradictory and heretical: apoptosis could not be pro-inflammatory since its primary function was to silently eliminate the cells, allowing for tissue remodelling during development. The magic of semantics: we simply had to rename this phenomenon “pyroptosis” for the concept to be accepted! With death and signalling we were at the heart of the concept of infectious danger perception.

Figure 3a. Entrance point of Shigella by macropinocytosis in an epithelial cell.

Figure 3a. Entrance point of Shigella by macropinocytosis in an epithelial cell.

Scanning electron microscopy.

Figure 3b. Shigella’s actin-dependent intracellular motility

Figure 3b. Shigella’s actin-dependent intracellular motility

Electron Microscopy Platform, Pasteur Institute.

26The transition between molecular genetics, cellular biology, and immunology was in any case guaranteed, as the infected cell provided an excellent common ground for multidisciplinary studies. Cellular microbiology was born during this time, inspired in our case by Patrice Boquet’s pertinent advice. A microbe-cell interface soon proved very fruitful, particularly at the Pasteur Institute, in other models like that of Listeria which was remarkably well analyzed by Pascale Cossart’s group. The 1990s saw the creation of the Microbial Molecular Pathogenesis Unit, then the INSERM Unit. Excellent young scientists put their trust in me: Claude Parsot, who, with Robert Ménard and Abdel Allaoui, developed the concept of a Type-3 secretion system; Guy Tran Van Nhieu, who deciphered the foundations of the molecular signalling of Shigella’s entrance into cells; Armelle Phalipon who outlined the host’s immune response against Shigella; and, last but not least, Nancy Guillen, who had the wisdom to choose another enteroinvasive pathogen, Entamoeba histolytica, the pathogenic amoeba; and Régis Tournebize, who chose to work on Klebsiella and pulmonary infection. Diversity first.

27I will attempt to share with you the main stages of this “Shigella saga”. The virulence plasmid holds a coding pathogenicity island for a molecular syringe or Type-3 secretion system, capable of injecting into the cellular membrane two proteins IpaB and IpaC that form a translocator capable of injecting other molecular effectors into the cell cytoplasm. Once in the cell, the bacterium breaks through the vacuolar membrane and escapes into the cytoplasm, where a movement is triggered allowing for the shift from cell to cell through protrusions; hence the rapid “underground” colonization, protecting the bacterium against the host’s defences. These two phenomena, entrance and motility, have become two paradigms of microbial pathogenicity. Their analysis has also contributed to the rapid development of the dynamic cellular imaging of host-microbe interaction. In this domain, observation often precedes discovery and stimulates it. That is why this year my series of lectures will conclude with an international symposium on in vitro and in vivo infectious processes, titled “Seeing is Believing”.

  • 10 Arp2/3: protein with 7 sub-units regulating the nucleation and assemblage of the cell’s actin filam (...)

28Entrance is linked to the capacity of the C-terminal domain of the IpaC invasin, now rooted in the cell membrane, to activate the small GTPases of the Rho family, thereby causing the nucleation of actin polymerization foci through the Arp2/3 complex. This is however not enough for an effective focus to form. It is the c-src proto-oncogene, recruited and activated by another sequence of the IpaC’s C-terminal domain, that phosphorylates cortactin, which itself causes the massive assemblage of actin filaments through the Arp2/3 nucleator complex10, remotely from the bacterial body. The latter can then be effectively internalized through macropincytosis. Each of these steps can be imaged: the secretion of Ipa proteins (thanks to Jost Enninga’s work), the rearrangement of the cytoskeleton, the recruitment of a key signalling molecule like c-src.

  • 11 N-WASP: protein with an acidic domain that recruits and activates Arp2/3.

29Motility is linked to the anchoring on the external bacterial membrane of an autotransporter protein, IcsA, which recruits the N-WASP molecule11 and activates it, thus leading to the nucleation of actin through the Arp2/3 complex, as the bacterium moves within the cytoplasm at the speed of this polymerization process. Here again, intracellular motility and the movement from cell to cell can be imaged. Pathogenic bacteria learnt about cellular biology before us.

30Yet one enigma remained: Shigella is very ineffective in entering through the intestinal epithelium’s apical pole. The combination of in vitro and in vivo approaches has allowed us to show the crucial role played by the M cells of the specialized epithelium covering the lymphoid follicles associated with the intestinal mucus. Although it is easy to cross through these translocator cells, the subepithelial dome is very rich in macrophages, thus creating a deleterious environment for bacteria. This is where the macrophage pyroptosis comes in, allowing Shigella to survive and invade the basolateral pole of epithelial cells, but also to trigger an inflammatory process destabilizing the epithelial barrier, thereby facilitating its invasion.

31The following step is a clear illustration of the educational potential of these microorganisms. Thierry Pédron and I had observed that in an intracellular position Shigella was able to reprogramme the epithelial cell, causing it to express pro-inflammatory genes. This observation led Dana Philpott and I to discover that a group of proteins with Leucine-rich sequences and that fix in the GTP, the Nod proteins, were intracytoplasmic bacteria sensors and that their microbial agonist was muropeptide, a fragment of peptidoglycan, the major component of the bacterial wall. Around the same time, Jean-Pierre Hugot and Gilles Thomas showed that the aggressive familial forms of Crohn’s disease were often associated with a mutation in the Nod2 gene. We immediately joined forces and demonstrated that these mutations led to a loss of function by Nod2 in the presence of its agonist, muramyl dipeptide. And yet how could a Nod protein, in the presence of a muropeptide, cause a severe inflammation during a Shigella infection and likewise cause a deleterious inflammation when, muted, it becomes non-functional in Crohn’s disease? This paradox takes us back to the tolerance to commensals/response to pathogens duality. We will discuss this in one of my lectures.

  • 12 NALP: family of proteins whose role is to organize the cellular response to different environmental (...)

32We had thus identified a new key element of the microbial danger perception system: the untimely and uncontrolled irruption of foreign bacterial molecules in the cell cytoplasm, seen as evidence of an aggression by a pathogen. It is in this type of situation that the boundary between commensals and pathogens becomes clearer. It was recently discovered that a complex network is involved, including for example helicase proteins like Rig-1, capable of sensing not only the intracellular presence of double-stranded viral RNA, but also molecules like the NALP12, able to detect and signal more generic elements of danger that are not necessarily microbial: potassium leak, increased ATP, uric acid crystals.

33This pro-inflammatory reprogramming of the epithelium by Shigella causes the subversion of the epithelial barrier, thus facilitating access and invasion by the pathogen. It also leads to the activation of powerful antimicrobial mechanisms: antimicrobial cationic peptides expressed and secreted by the epithelium, recruitment and transepithelial migration of polynuclear neutrophils and dendritic cells (Figure 4).

Figure 4. Diagram showing the rupture, invasion and inflammatory destruction of the intestinal epithelium by Shigella.

Figure 4. Diagram showing the rupture, invasion and inflammatory destruction of the intestinal epithelium by Shigella.

34It is therefore not surprising to observe that Shigella injects into the cells, through the Type-3 secretion system, a series of effectors encoded by the virulence plasmid: the Osp and IpaH proteins, which are enzymes that attack key components of pro-inflammatory signalling. Let us not be afraid to talk about toxins anymore. Dong Wook Kim and Claude Parsot have shown that the OspG protein is anti-inflammatory, as it links itself to the ubiquitinylated form of the E2 enzymes which transfer the ubiquitin involved in the deterioration of IkappaB, the chaperon that keeps the pro-inflammatory transcription complex NF-kappaB inactive in the cytoplasm. Laurence Arbibe has shown that the OspF protein is concentrated in the core of the infected cell, where it dephosphorylates the MAP kinases and, in so doing, triggers the dephosphorylation of the histone H3’s N-terminal tail. This ensures the heterochromatinization, and therefore the inhibition, of a number of pro-inflammatory gene promoters, like IL-8, a powerful polynuclear neutrophil chemoattractant. The epigenetic regulation of inflammation by a toxin is the work of bacteria! Cell nucleus biology therefore finds its way into our research topics, especially since OspF drastically regulates the transepithelial migration of polynuclear neutrophils.

35Brice Spérandio, Thierry Pédron and I recently showed that these injected effectors suppress the expression of antimicrobial peptides and the recruitment of dendritic cells, creating optimum conditions for the colonization of the mucus.

36Pro-inflammatory and anti-inflammatory Shigella are the Yin and the Yang of the innate immunity controlled by bacteria: more surprises are yet to come. And we thought only viruses were intelligent!

  • 13 Secretory IgAs: antibody isotype that can be secreted through the mucosal epithelium.

37I have not spoken about adaptive immunity yet. Armelle Phalipon demonstrated that secretory IgAs13 are a major component of protection against Shigella on two levels: the immune exclusion of microorganisms, which cannot interact effectively with the epithelium, and the reduction of epithelial inflammation through the interception of bacterial components like lipopolysaccharide in the apical recycling compartment of the epithelial cell. She was recently able to show that Osp proteins also regulate adaptive response. The next step is to move away from our chronic “epitheliocentrism” and to look at the effect of the injection of Shigella effectors into immune cells, especially antigen-presenting dendritic cells, and B and T lymphocytes. The gamma interferon is a crucial mediator of the innate and adaptive anti-Shigella response; there is a growing body of evidence of the negative regulation of this cytokine by the pathogen.

38This saga has led not only to the deciphering of Shigella’s genetic and molecular identity as a pathogen, but also to the development of more general systems and concepts in the domain of infectious and inflammatory diseases. In the Pasteurian tradition, we have sought to draw useful applications from this research. We are pursuing a programme to develop an oral vaccine against bacillary dysentery. A series of selected genetically attenuated strains has been built, displaying both tolerance and immunogenicity, in order to develop a live vaccine that can easily be administered orally in endemic regions. The central mutation concerns the icsA gene. Once this gene has been deleted, the bacteria loses its capacity to move from cell to cell, and therefore to colonize the entire epithelium. We previously showed the absence of clinical symptoms in the macaque monkey following the oral administration of these potential vaccines and an epithelial crossing at the level of the lymphoid structures associated with the colorectal mucosa: an ideal scenario for an oral vaccine, leading in particular to the production of protective secretory IgAs. These potential vaccines have been and still are undergoing clinical trials on humans, initially at Walter Reed, at Fort Detrick and at the St Georges Vaccine Institute in London, and more recently by Christine Sadorge and Marie-Lise Gougeon at the Pasteur Institute, and Odile Launay at the Cochin-Pasteur Vaccinology Centre. Phase-3 trials now need to be prepared, to test its effectiveness in endemic regions. With this goal in mind, Yves Germani and I are implementing an integrated programme to study serious paediatric enteric diseases in endemic zones through the international network of Pasteur Institutes. This includes the development of rapid diagnostic tests of enteric pathogens through immune-chromatography on a medium directly exposed to the pathological material, the epidemiological analysis of the disease’s incidence and its impact on children’s health, and the identification of sites to carry out the vaccine’s Phase-3 test in the future.

39But we have other irons in the fire. Armelle Phalipon, in collaboration with Laurence Mulard, is developing a highly innovative approach with a parenteral vaccine involving the chemical synthesis of the complex polysaccharide antigens of the lipopolysaccharide at the surface of Shigella, known to be its dominant protective antigen. We are developing other approaches with industrial partners, the tide is turning... Shigellosis has gone from the rather undesirable status of an ignored disease to the very envied status of a neglected disease.

The emergence of infectious diseases: from fatality to a proactive approach and anticipation

40Ultimately, is the emergence of infectious diseases fatal? “There will therefore be new diseases”, said Charles Nicolle. “That is a fatality. Another fact, equally fatal, is that we will never be able to track them down from their origin. When we become aware of these diseases, they will already be completely formed, adult, one could say. How will we recognize these new diseases, how will we be able to suspect their existence before they manifest themselves as symptoms?” What has become of these predictions made before the introduction of anti-infectious agents and of most vaccines? They have been fully confirmed.

41A recent article in Nature by Kate Jones and her colleague inventories the appearance of 335 infectious emergences between 1940 and 2004, including about 100 in the 1980s, largely related to Aids immunodeficiency. Most were recorded in high latitude regions, most likely reflecting the lack of identification and reporting in countries of the South. There is a predominance of zoonoses, three quarters of which are transmitted by wildlife (HIV, Lassa, Marburg, Ebola, SARS). Most have a bacterial etiology, due to the bias in publicizing emergences related to the acquisition of resistance to antibiotics.

42Looking at the future, the conditions of emergence will remain the same, predominantly stemming from socio-economic, environmental and ecological factors. This is especially true of industrialized countries: dangers of the industrial food chain and the development of increasingly complex environments like large complexes’ ventilation systems, a safe haven for Legionella pneumophila, and hospital environments, which combine nosocomial risk with the risk of multiresistance to antibiotics. The global methicillin-resistant staphylococci pandemic, the appearance of vancomycin resistance first in enterococci, and now in staphylococci, provide examples of constant emergences with astronomical societal costs. The planet’s underprivileged regions – two billion deprived people live with less than a dollar a day – will bear the brunt of infectious emergence in the future. Risk factors involve the steady growth of the population until 2050 at least, the growing concentration of populations deprived of access to education and hygiene on the outskirts of megacities, and the growing invasion of forest zones, increasing the chances of encounters with new vectors and wild animals, reservoirs of infectious agents. Furthermore, the poorly controlled introduction of an industrial food chain and the development of hospital structures with poorly managed standards in economically emerging countries encourages the development, on a scale we have not experienced in our regions, of food and nosocomial infections by multi-resistant germs.

43The concept of a North-South boundary in the appearance of emerging infections is absurd, even dangerous. The continuous increase in travel and exchanges (2.1 billion air passengers in 2006) has turned the Earth into a global village where infectious agents circulate as freely as do people. It did not take long for the SARS virus to jump from a remote province of China to Hong Kong, then Toronto.

44If an additional factor of infectious emergence risk for the future had to be given, it would relate to the general ageing of populations. In 2050, close to a third of the European population will be over 65. This trend towards ageing is just as real in emerging countries. The incidence of infectious diseases massively increases at the two extremities of the life cycle, where the effectiveness of vaccinations declines. With age, lymphopoiesis wanes, the quality of activation signals deteriorates, and especially the thymus – which teaches lymphocytes to fulfil their function of new antigen recognition – involutes. The pool of lymphocytes likely to be involved in the recognition of new antigens, and therefore new infectious agents, collapses and the few cells available are depleted fighting chronic viral infections like those linked to the viruses of the Herpes family. If the latter mechanism were confirmed, the development and use of vaccines against infections would become a priority: vaccination as prevention against the immune system’s senescence is a new paradigm to consider. It is urgent to think about delaying the immune system’s retirement age.

45It will be possible to control infectious diseases only if we manage to create the conditions for a real “sanitary transition” on a global scale. This intricate concept involves requirements that vary, from discovering innovative anti-infectives, to developing vaccines against major endemic diseases, to improving sanitary conditions in suburban ghettos, to strengthening emergence monitoring and rapid response networks, reoriented towards the South. Internet probably saved us from SARS. The time has also come for forward-looking monitoring in areas at risk of microorganism transmission from wildlife to humans. Antoine Gessain at the Pasteur Institute will give a seminar on this molecular monitoring of the movement of retroviruses in Africa. Finally, a sustainable economic model needs to be consolidated, taking infectious diseases into account. The public-private partnerships that have been initiated over the last few years, including by large foundations, are undeniable progress affording support for targeted disease control projects. But in the long term it is the responsibility of States to pursue this struggle through education and prevention. Although the means needed may seem tremendous, they are nothing in comparison to the cost of an epidemic, or a pandemic: SARS cost 60 billion dollars to Asian countries’ economies. And who will ever be able to calculate the real human and material cost of the Aids pandemics?

Research, the winning bet for controlling infectious diseases

46Research is the best investment we can offer: microbiology, both human and veterinary, one and indivisible, physiopathology and immunology, epidemiology and the humanities, information technology and the modelling of epidemics, new diagnostic tool engineering, development of new medicines and vaccines. When he made his red queen say to Alice that “It takes all the running you can to keep in the same place”, Lewis Carroll was brilliantly summing up our situation as we face the microbial world whose diversity and evolutionary speed allow it to adapt to all the situations we seek to impose on it. If we do not move forward, the regression will be fatal. Allow me to cite here the introduction to the Académie des sciences’ “Science and Technology” report on controlling infectious diseases, of which Gérard Orth and I were recently the editors: “Fundamental research ensures the acquisition of a knowledge base whose quality and diversity should easily allow a state of preparedness. Infectious diseases offer fundamental research excellent paradigms of integrated and trans-disciplinary approaches to study complex biological processes. Research is what will make it possible to reduce the margin of uncertainty that exists in the appreciation of infectious risk and on which the application of the precautionary principle is based.”

47Controlling infectious diseases is a complex and intricate activity. Take the case of vaccines. There are three crucial commandments:

48Do not let the guard down. In the early 1970s in the United Kingdom, following a virulent campaign against the anti-pertussis vaccine which was perceived to be poorly tolerated, the country’s vaccine coverage collapsed: the incidence of pertussis, then close to zero, suddenly began to rise again. Controlling the resurgence was only made possible by adequate vaccine coverage, using vaccines that were better tolerated. This experience and the current return of pertussis in young adults show us that it is only by maintaining a life-long vaccine coverage that a disease can be controlled. The eradication of smallpox was made possible by a certain number of characteristics of the disease and its virus, which are not necessarily found with other pathogens that persist in our environment and re-emerge as soon as we let our guard down.

49Do not give up. In 2000, the world was faced with a medically and ethically unacceptable situation: one million children in deprived countries were dying each year from measles or developed poliomyelitis, even though very effective vaccines were available. Owing to an international effort coordinated by the WHO, involving strong public-private partnerships, this figure was brought down to 250,000 in 2006, offering hope that these diseases could be controlled. A proactive approach pays off in public health.

50Do not reduce research efforts. Take the example of vaccination against meningococcal meningitis C. An extraordinary academic and industrial research effort has allowed for “polysaccharide conjugated” vaccines that have proven very effective on capsulated bacteria like Neisseria meningitides, Streptococcus pneumoniae and Hemophilus influenza, to become available on the market. The United Kingdom was able to control its meningococcal meningitis C endemics thanks to this type of vaccine, beyond what it had hoped for. Epidemiological data reveal a zero incidence rate even before the date forecast by the most optimistic projections. This is due to the unexpected fact that these vaccines not only protect against the disease but also considerably reduce the carriage and transmission of the causative agent hence the new concept of “herd protection”. These vaccines stemming from research are outperforming nature. While the commercialization of new vaccines is actually continuing at a sustained rate, many shortfalls and needs are still felt with new concepts of vaccines and adjuvants for complex pathogens like Plasmodium, BK and HIV. Only fundamental immunology and microbiology will be in a position to provide effective contributions.

51One last word, of optimism, on the extraordinary impact of research on the control of infectious diseases. Here are three examples:

  • The generalization by the French Blood Establishment of the Viral Genomic Diagnosis, which is a pure product of research (PCR, discovery and sequencing of the genome of the viruses concerned), has afforded transfusion security in France, on an unprecedented scale.

  • The domain of anti-virals, which was characterized by the rarity of effective and non-toxic medicine, recently saw new and very innovative molecules, active on a broader range of viruses, made available thanks to a considerable research effort, partly stimulated by the explosion of Aids. This domain has in fact been more prolific than that of anti-bacterials, antifungals and anti-parasites, for which innovation needs remain huge.

  • Finally, the discovery of HIV at the Pasteur Institute (which has just been honoured by the Nobel Prize awarded to Françoise Barré and Luc Montagnier) and the fundamental research on retroviral biology which immediately followed (including its sequencing) are what allowed for the development of diagnostic tests and treatments to control the disease. Rootedness in medicine, fostering of extensive knowledge of the microbial world, and capacity for rapid mobilization of fundamental science and the means to support it are crucial parameters of success. Louis Pasteur left us with the keys to these through a reactive organizational model of biomedical research of time-transcending modernity. His model also involves support for applied research, rebaptized “translational”, which seeks to convert the phenomenal mass of fundamental knowledge into useful tools to control infectious and parasitic diseases. This is a human, economic, and ethical requirement.

52I would like to express my immense gratitude to the colleagues I was not able to mention, particularly my students and post-docs, to those who taught me medicine and science, to my family, friends and colleagues who are here with us today, to my mother and my father who is no longer here, and to Nicole, my wife.

53I will leave you with Charles Nicolle, to whom I humbly dedicate this lecture: “Knowledge of infectious diseases teaches men that they are brothers and all bound together. We are brothers because the same danger threatens us all, and bound together because contagion often reaches us through our fellow humans”.

54And with Joshua Lederberg, who recently passed away. This global visionary of the microbial world, Nobel Prize Laureate at the age of 33 for the discovery, at 22, of bacterial genetic recombination, was an adviser to NASA on microbiology: “We live in evolutionary competition with microbes, bacteria and viruses – there is no certainty that we will be the winners.” My intention is certainly not to dampen the message of optimism with which I wanted to conclude this inaugural lecture! I think that Josh simply wanted to reassure us about the fact that, the day we all eventually come to our end, microbes will take care of us with professionalism and effectiveness.

55Thank you.


1 Human papillomavirus. This virus is responsible for a very large number of sexually transmitted disease and cervical cancers.

2 François Jacob, Le Jeu des possibles. Essai sur la diversité du vivant, Paris, Fayard, 1981.

3 Charles Nicolle, Destin des maladies infectieuses, Paris, Félix Alcan, 1933.

4 The virus responsible for bird flu.

5 Tolerogenic: which creates a state of immune tolerance to an antigen or to a microorganism.

6 PAMPs: pathogen-associated molecular patterns, molecules belonging to the prokaryotic world (lipopolysaccharide, peptidoglycan, flagellin, etc.), whose recognition allows the host to discriminate between the prokaryotic and eukaryotic worlds.
toll-like receptors, molecules involved in the perception of PAMPs.
DC: dendritic cells;
MΦ: macrophages.
Treg: regulatory T lymphocytes.

7 NLR : nod-like receptors, cytoplasmic sensors.

8 Plasmid: genetic element independent from the bacterial chromosome, capable of replicating autonomously, or even transferring.

9 Apoptosis: programmed cellular death which, unlike necrosis, involves not cell lysis but its fragmentation.

10 Arp2/3: protein with 7 sub-units regulating the nucleation and assemblage of the cell’s actin filaments.

11 N-WASP: protein with an acidic domain that recruits and activates Arp2/3.

12 NALP: family of proteins whose role is to organize the cellular response to different environmental stimuli, including certain bacterial compounds. The cell responds by liberating interleukin-1, thereby stimulating a cascade of mediators and of clinical inflammatory manifestations.

13 Secretory IgAs: antibody isotype that can be secreted through the mucosal epithelium.

Table des illustrations

Titre Figure 1. Mechanisms involved in the evolution of bacterial genomes
Fichier image/png, 67k
Titre Figure 2a. Commensalism6
Légende Commensals: Absence (limitation) of virulence factors. Less agonist PAMPs (not constant). Sequestration, less TLR activity. Life in biofilms on the surface of the mucus. Controlled dissemination and sampling of the PAMPs and prokaryotic signalling molecules.
Fichier image/png, 271k
Titre Figure 2b. Pathogenicity7.
Légende Pathogens: Mucinases. Adhesins. Invasins. Type III/IV secretion systems. Hemolysins Massive receptor engagement by microbial products.
Fichier image/png, 191k
Titre Figure 3a. Entrance point of Shigella by macropinocytosis in an epithelial cell.
Légende Scanning electron microscopy.
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Titre Figure 3b. Shigella’s actin-dependent intracellular motility
Crédits Electron Microscopy Platform, Pasteur Institute.
Fichier image/png, 356k
Titre Figure 4. Diagram showing the rupture, invasion and inflammatory destruction of the intestinal epithelium by Shigella.
Fichier image/png, 428k

© Collège de France, 2013

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