Medical Microbiology

The Parasites (Pathogens) Way of Life

Kelly Doran BIOL 584 Spring 12

Schaechter ch 3,8

Why do bacterial pathogens produce disease?

Factors that promote interaction with the host ! ! ! ! Adherence to host cells Invasion of host cells Competition for iron and nutrients Resistance to phagocytes ! Avoid complement and antibody ! Resist phagocytic engulfment ! Survive intracellular killing mechanisms

Host-Pathogen Interactions
!! Pathogenicity
"!Measure
!! Determined
"! Number

of organism

of ability to cause disease

of organisms to colonize 50% of hosts

by genetic makeup of organism !! Infectious dose = ID50

"!Virulence
!! Rate

of lethal infections !! Lethal dose = LD50

"! Number

of organisms to kill 50% of hosts

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Microbiology: An Evolving Science 2009 W. W. Norton & Company, Inc.

Infection Cycle
! Direct contact ! Indirect contact
! Contact with fomites ! Horizontal transmission via vectors
! MosquitoesYellow fever, malaria ! Reservoir for disease organism
! May not show disease symptoms

! Mode of entry depends on pathogen

! Mucosal surfaces, wounds, insect bites
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Virulence Factors - Microbial Virulence genes Toolkits

! Encode factors allowing pathogen to invade host
! Toxins, attachment proteins, capsules

! Pathogenicity islands
! Section of genome

! Contain multiple virulence genes

! Transferred as a block from other organisms

! Often encode related functions ! E.g., protein secretion system, toxin production

! Often flanked by phage or plasmid genes ! Often have GC content different from rest of genome

Bacterial Cell Architecture

The major structural components of bacteria include: - the double-stranded DNA genome -! cytoplasm containing ribosomes -! cell membrane - cell wall* and capsule* -!pili/fimbriae* and flagella* - spores*
*not in all bacteria
http://www.cellsalive.com!

Bacterial Surface

The surface of Bacillus anthracis. From Mesnage, et al. Journal of Bacteriology (1998) 180, 52-58. The bacterial membrane is evident as the innermost layer surrounding the cytoplasm.

Bacterial Cell Wall

Bacteria are classified into two major groups: Grampositive or Gram-negative following staining. Gram stain results reflect the thickness of the peptidoglycan layer in the bacterial cell wall.

Staphylococcus aureus

Escherichia coli

www.cat.cc.md.us!

BACTERIAL CELL WALL STRUCTURE

The structure of the cell wall:

www.cat.cc.md.us!

BACTERIAL CELL WALL STRUCTURE

The structure of the cell wall:

www.cat.cc.md.us!

Flagella

Vibrio cholerae. Liefson's flagellar stain (CDC). Vibrio cholerae is motile by means of a single polar flagellum inserted into one pole of the cell. Peritrichous flagella =

E. coli

Bacterial Pili Structure

Bacterial pili (also known as fimbriae) surface protein filaments involved in attachment to surfaces or to other bacteria.
Neisseria gonorrhoeae

Conjugation

http://textbookofbacteriology.net!

Pili, coined by Charles Brinton (Nature 1959), comes from the Latin for hair or fur. Fimbriae, coined by James Duguid (J. Pathol. Bacteriol. 1955), derived from the Latin word for fringe.

Bacterial Pili/fimbriae: hair-like structures 5-7nm diameter with varied morphology. structurally diverse and different fimbriae recognise different host receptors.

Bald E. coli

Bundle-forming pili (enteropathogenic E. coli - EPEC)

CS1-piliated enterotoxigenic E. coli (ETEC)

J. Bacteriol. 1996 178:65556563!

Gram-negative Pili
! Shaft - repeating protein subunits (pilins) assembled into a helical array ! Adhesive subunit(s) ! Specialised tip structure (Pap pili UPEC) ! Tip no specialised structure (N. meningitidis). Adhesive subunits along length of shaft

Pap (pyelonephritis associated pili) associated with uropathogenic E. coli (UPEC) which causes UTIs (bladder, kidney infections).......

Gram-positive Pili in Streptococcus

pilA

pilB

pilC

Maisey, Doran J. Bacteriol. (2007)

Bacterial Capsule Structure

Many bacteria produce a capsule, which usually consists of a sugar polymer. The capsule may play different roles in different bacterial Genera: - prevent desiccation - facilitate adhesion to surfaces - prevent phagocytosis -!an external carbohydrate reservoir
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Capsules

Colonies of Bacillus anthracis.

The slimy or mucoid appearance of a bacterial colony is usually evidence of capsule production. The B. anthracis capsule is composed of poly-Dglutamate and is an essential determinant of virulence to the bacterium. In the early stages of colonization and infection the capsule protects the bacteria from assaults by the immune and phagocytic systems.

The Capsule Polysaccharide of Group B streptococcus (GBS)

! Almost all GBS are encapsulated belonging to one of 9 serotypes
!4" !4" !4" !4" ! Sialylated capsule acts as a primary !3" !6" !3" bloodstream survival factor #3" !4"

WT

Ia!

#3"

Ib!

V!

!3"

#3"

!4"

! Promotes bacteremia by inhibiting !4" !4" !3" !4" !3" !2" activation of the alternative pathway !6" !3" !6" #3" II! of complement, interfering with VII! !4" neutrophil phagocytosis
#3" !6" !3" !4"

Mutant!

Glucosamine CPS mutants have diminished Galactose #3" glucose Sialic acid!in animal models virulence

III!

Bacterial Spores
Bacterial spores survive dessication and heating. Spores germinate to form vegetative cells eg. Bacillus anthracis. Vegetative cells usually are destroyed at 60oC while spores require heating to 121oC for 15 min.

Knox, 3rd edition 2005!

Bacterial Spores

Microbial Attachment
! Human body expels invaders
! ! ! ! Mucosa, dead skin constantly expelled Liquid expelled from bladder Coughing, cilia in lungs Expulsion of intestinal contents

! Bacteria must adhere to host tissue

! Pili (fimbriae)
! Afimbrial

! Adhesinssurface proteins bind host cells

! Hollow fibrils with tips to bind host cells

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Mucin Layer in Intestine

Microbial Adherence to Host Epithelium

Adherence to skin or mucosal surfaces is an fundamental characteristic of the normal human microflora

Mucosal adherence is also an essential first step in the pathogenesis of many important infectious diseases

Adhesins:

Microbial Proteins that Mediate Adhesion to Host Cells

adhesins in the! bacterial cell wall!

! Direct adherence interactions: (surface glycolipids,glycoproteins, glycosaminoglycans) ! Indirect adherence interactions: (matrix glycoproteins, mucin) ! Tissue tropism is determined by the array of adhesinreceptor pairs

host cell membrane

adhesin! receptor

Bacterial adhesins:
surface structures that bind to specific host receptors:
! fimbrial/pilus adhesins ! afimbrial adhesins

! overcome electrostatic repulsion (bacterial and host cells are both negatively charged

Bacterial Pili

Telford et al. Nature Reviews Microbiology 4, 509519 (July 2006) | doi:10.1038/nrmicro1443

Biofilm Development
Stages:

Biofilms
! Cells secrete material to hold to a surface
! Cells acting together
! Multiple species or a single species ! Quorum sensing

! Cells signal to each other ! Protects against dispersion ! Prevents antibiotics from infiltrating

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Afimbrial adhesins
Gram-positive afimbrial adhesins eg. streptococcal fibronectin binding proteins of Streptococcus pyogenes binds the ECM component fibronectin.
Confocal microscopy image of fibronectin (stained green) as part of the extracellular matrix surrounding cells (nuclei stained blue). Fibronectin binds to the human cell surface via the #5!1 integrin receptor.

ECM Adhesion

Figure 1:(A) Fibronectin binding by Bacillus subtilis. (B) Specific fibronectin binding by surface proteins of Group A Streptococcus.!

ECM Adhesion
SfbI mediates uptake of S. pyogenes into human cells by binding to fibronectin attached to the #5!1 integrin receptor, via a complex 4 step process.

#5! 1 integri n

Fibronectin

RGD

I
S. pyogenes

III

IV
SfbI protein

II

Pili/Fimbriae! Intimin!
Pedestal! Host !-integrin! Host glycolipid! or glycoprotein! Host cell surface! protein/carbohydrate! Host cell ! membrane!

Major subunit! Tip (pili)! adhesin !

Actin! polymerization!

P!

Secreted ! Hp 90 !

Ambrial! adhesins!

Bacterial Intracellular Invasion

Why would bacteria want to invade/enter a host cell?

Invasion

Streptococcus

Anthrax Staphylococcus

Salmonella Invasion
Triggering by Type three secretion systems (TTSS) !

Actin polymerization # cup-like ! structure! "Actin depolymerization # uptake !

Type III Protein Secretion

! Injects proteins directly into host cell ! Injected proteins cause host to engulf bacterium
! Hypodermic needle similar to base of flagellum! ! Genes on pathogenicity island

! Salmonella injects over 13 toxins

! Alters fusion of vesicles in cell ! Causes diarrhea in host

TTSS Molecular Syringe

Bacterial Invasion & Molecular Switches

Salmonella SopE

Pseudomonas ExoS, Salmonella SptP

Functions of Rho GTPases

! Affect the architecture of the actin cytoskeleton. ! The three main subgroups encompassing the Rho family, Rho, Rac and Cdc42, transduce extracellular signals into changes of the actin cytoskeleton ! Required for the formation of stress fibers, membrane ruffles and filopodia, phagocytosis

Rho GTPases activation/ deactivation cycle

Inactive

Active

Opsonization by complement (C3b) " or specic antibody greatly" enhances phagocytosis

Streptococcus pyogenes

Inhibition of phagocytosis

Interference with Antibody Binding and Opsonization

Staphylococcus aureus Protein A

Bacterial Intracellular Invasion

Other bacterial pathogens able to invade and survive within host cells include:
! Listeria monocytogenes ! Mycobacterium tuberculosis ! Mycobacterium leprae ! Shigella dysenteriae
Listeria monocytogenes moves within and between cells by polymerizing actin.
eaton.math.rpi.edu!

! Once inside host cell, how to avoid death?

! Cell ingests pathogens in phagosome
! Some pathogens use hemolysin to break out
! Shigella dysenteriae, Listeria monocytogenes

Surviving within the Host

! Phagosome fuses with acidic lysosome

! Some pathogens secrete proteins to prevent fusion ! Some pathogens mature in acidic environment
! Coxiella burnetiiQ fever ! Salmonella, Chlamydia, Mycobacterium, Legionella

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Intracellular Survival
Bacteria Macrophage or neutrophil

Antimicrobial Peptides (AMPs)

(Defensins, cathelicidins)

AMP Resistance Mechanisms

ROS

Bacterial Defense

54

Iron acquisition
siderophores

Siderophore Receptors