Interactions between symbiotic microbes, their mammalian host, and invading pathogens

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1 Interactions between symbiotic microbes, their mammalian host, and invading pathogens Vanessa Sperandio UT Southwestern Medical Center Depts. Microbiology and Biochemistry

2 To have a quorum you need chemicals

3 Bacterial density and diversity Human gut is a complex and highly populated environment: trillions of commensal bacteria Intestinal microbiota is abundant and diverse (estimated species) Small intestine 10 7 CFU/mL Large intestine CFU/mL Firmicutes and Bacteroidetes are predominant Chemical signaling in the GI tract helps to coordinate population behavior Carbon and utilization capabilities play a major role for homeostasis of commensal bacteria and infection by pathogenic species

4 Host-microbial interactions Human cells Gastrointestinal tract bacterial cells Indigenous microbiota plays an important role in nutrient assimilation and production of vitamins An important role in the development of the mammalian digestive and immune systems Microbes modulate expression of mammalian genes Inter-kingdom chemical signaling between microbes and host Signaling can be exploited by pathogens

5 Bacterial-host associations Pathogenic bacteria: Cause disease in the host Opportunistic bacteria: Do not cause disease in healthy individuals. Cause disease in a susceptible (e.g. immune-suppressed) host. Normal flora: do not cause disease and live in symbiosis (colon, skin, oral cavity). Aid in the digestion of food, production of vitamins and protects the host from colonization by pathogenic organisms

6 E. coli pangenome Comparison of 17 E. coli genomes David Rasko Includes a true E. coli commensal Isolated from a healthy scientist Between all E. coli genomes Only 2,200 genes are shared (E. coli genomes vary between 4,000 to 5,000 genes) Pangenome of E. coli has a gene Repertoire of 13,000 Rasko et al. J. Bacteriol. 2008

7 Enterohemorrhagic E. coli (EHEC) O157:H7 Responsible for outbreaks of bloody diarrhea in many countries Infection by contaminated food and water Low Infection dose ( cfu) Colonizes the large intestine Higher probability of developing Hemolytic Uremic Syndrome (HUS) in the young and elderly.

8 lumen EHEC Commensal bacteria live in the outer loose mucus layer and present serious competition for enteric pathogens Outer loose mucus layer Commensal bacteria inner firmly attached mucus layer CV Intestinal epithelium AI-3 Epi/NE

9 Locus of Enterocyte Effacement (LEE) McDaniel et al, 1995 T3SS of EHEC Deng et al; PNAS, IRTKS EspFu N-WASP Arp2/3 Actin Garmendia et al., Infect Immun

10 Charley Gruber

11 However, expression of this type III secretion system is expensive for EHEC. It needs to be sure when, where, how much Abe et al. PNAS 2001 Abe et al. PNAS 2001 Knutton et al. EMBO 1998

12 lumen EHEC Commensal bacteria live in the outer loose mucus layer and present serious competition for enteric pathogens Outer loose mucus layer Commensal bacteria inner firmly attached mucus layer CV Intestinal epithelium AI-3 Epi/NE

13 Walters et al. J. Bacteriol I V. harveyi Epinephrine AI-3/epi/NE inter-kingdom signaling Tyrosine Norepinephrine AI-3 (What s known) Epi/NE Host signals Epinephrine and norepinephrine are the stress fight or flight hormones NE/Epi control intestinal motility Cl - and K + secretion NE produced in the intestine by the ENS NE/Epi control the immune system

14 How does EHEC sense Epi/NE? Sensed by GPCR in mammalian systems E. Coli has no GPCRs

15 QseC QseC is an inner membrane bacterial adrenergic receptor that responds to the bacterial signal AI-3 and to the host signals epinephrine and norepinephrine. Signaling of QseC leads to autophosphorylation and subsequent phosphotransfer to QseB, QseF and KdpE David Hughes AI-3 N Epi TM1 TM2 P NE His Kinase EAL C ATPase QseC (present in all lanes) QseB Clarke et al. PNAS 2006 Rasko et al. Science 2008 Hughes et al. PLoS Pathogens 2009 QseB KdpE QdeF

16 H N TM1 TM2 C EAL His Kinase ATPase P QseB QseF KdpE NE Epi AI-3 N TM1 TM2 C EAL His Kinase ATPase N TM1 TM2 C EAL His Kinase ATPase TM1 TM2 C EAL His Kinase ATPase P QseB QseF KdpE QseB QseF KdpE NE Epi AI-3 NE Epi AI-3 B

17 QseC Epi/NE AI-3 P Flagella LEE Shiga TTSS toxin effectors

18 AI-3 PO4 Epi/NE Epi/NE SO4 David Rasko LEE AE lesion KdpE P QseC P QseB EspFu TccP P QseE QseF P SOS Marcie Clarke Marcie Clarke David Hughes Flagella Flagella motility qsee qseg qsef glnb Shiga toxin Nicola Reading Clarke et al PNAS; Rasko et al Science; Reading et al J. Bacteriol. Readin et al PNAS; Hughes et al submitted

19 Infant EHEC rabbit model EHEC colonizes the large intestine Causes diarrhea Established virulence mutants of EHEC in the LEE genes or Shiga toxin are attenuated in this model Matt Waldor Ritchie and Waldor (Brigham and Women s Hospital, Harvard Medical School) Rasko et al Science

20 Is the AI-3/Epi/NE signaling restricted to E. coli? QseC: EHEC, EPEC (causes diarrhea) UPEC (UTI infections) Shigela flexneri (dysentery) Salmonella sp. (typhoid fever) Yersinia pestis (plague) Y.enterocolitica, pseudotuberculosis(gastroenteritis) Erwinia carotovora (plant pathogen) Pasteurella multocida Haemophilus influenzae (meningitis, pneumonia) Actinobacillus pheupneumoniae P. aeruginosa (cystic fibrosis; burn patients)*** Burkholderia cepacia (cystic fibrosis) Coxiella burnetti (Q fever) Ralstonia solacenarum (plant pathogen) Francisella tularensis (tularemia) ** Vibrio cholerae (cholera) ** Vibrio vulnificus (gastroenteritis) Vibrio parahaemoliticus (gastroenteritis) Legionella pneumophila (Legionnaires disease) Bordetella pertussis (whopping cough)

21 Summary EHEC senses the flora derived QS signal AI-3 and host hormones epi/ne to recognize it is in the colon of the host to initiate infection The bacterial sensor for these signals are the sensor kinases QseC Initiates a complex phosphorelay cascade that regulates expression of virulence genes in vitro and in vivo

22 Regan Russell Meredith Curtis Cristiano Moreira Melissa Kendall Reed Peifer Christopher Parker Alline Pacheco Jacqueline Njoroge Charley Gruber Y. Nguyen Former members David Rasko Nicola Reading Marcie Clarke David T. Hughes Matthew Walters Marcie Clarke Fernanda Pace Marcelo Sircili Faith Sharp Shane Flickinger Juliana Falcao Sergio Rocha Carlos Esteban Nieto Ben Habdas Fernanda Franzin Acknowledgements Charley Cris Moreira Ben Alline Jacky Dave Hughes Harvard Matt Waldor and Jenny Ritchie UT Southwestern: Camille Falck Melissa Meredith Marcie Clarke Marcie Clarke Dave Rasko Chris Parker NINDS/ NIAID/ The Ellison Medical Foundation/ Burroughs Wellcome Fund High Risk High Impact grant UT Southwestern Regan Matt Nikki Y Nguyen