Microbial Pathogenesis

October 18, 2011by

David E. Brilesdbriles@uab.edu

934-6595

Whether a pathogen is able to cause symptoms (disease) is dependent on the interaction of the pathogen with the host.

This interaction is called Microbial Pathogenesis.

Bacterial Pathogenesis

Innate immunity & antigen-specific adaptive immunity

Microbial virulence mechanisms(mediated by virulence factors)

Protects against pathogens and tumors

Allows the pathogen to evade immunity, survive in the host, and spread to others

How do these opposing forces stay in balance?

Understanding Microbial Pathogenesis leads to:

• New anti-microbial drugs (antibiotics)• New and improved vaccines• Use of cytokines to stimulate innate

immunity• Better supportive therapy• New preventive measures

Disease Symptoms

• generally are the result of inflammation caused by host response to the pathogen.

• can be caused by toxins of the pathogen that damage the host.

• can be caused by chronic infection that leads to erosion and destruction of host tissue and sometimes whole organs.

What is the importance of disease symptoms?

Antibiotics• Originally from natural products (selected in nature

from the competition between microbes).

• Antibiotics block essential microbial-specific functions. (DNA replication, protein synthesis, cell wall synthesis etc.)

• Antibiotic resistance is the result of mutations and gene exchange which lead to new variants of the original pathogen.

• Future antibiotics will exploit new targets identified by studies of microbial pathogenesis.

In vivo studies of microbial pathogenesis are conducted by

• Administering of drugs, cytokines, antibodies or gene products that target specific suspected bacterial or host mechanisms.

• Genetic knock-out mutants in,– the host (innate immune factors, adaptive

immunity, cell receptors)– the pathogen (virulence factors)

Classification of Microbes (with respect to human health)

Adapted to Man Type Properties

No Does not colonize or infect man Free living or in other hosts

No Opportunistic Causes disease in immune compromised

a. infantsb. elderlyc. sick individualsd. immune deficiente. AIDS patients

Yes Normal flora Commensal or symbiotic-- cause no symptoms

Yes Disease Producing,Maintained by

a. colonizationb. infection

Can cause clinical symptoms & sometimes death

a. occasionally cause diseaseb. always cause disease

Normal FloraReside in:

gut, especially the large intestineoral cavityupper airwaysgenital tracturinary tractskin

Normal FloraCause no clinical symptomsAre often beneficial:

exclude pathogens by occupying available nichesproduce some vitamins (especially vitamin K)

Attributes of a Pathogenic Microbe

• Acquisition by a host• Exploitation of a host niche environment

– Usually this niche is in sterile tissue (otherwise it would generally not cause disease symptoms)

• Evasion of host defenses (usually partial)

• Multiplication in the host• Produce disease symptoms• Transmission to others

Attributes of a Pathogenic Microbe

• Acquisition by a host• Exploitation of a host niche environment

– Usually this niche is in sterile tissue (otherwise it would generally not cause disease symptoms)

• Evasion of host defenses (usually partial)

• Multiplication in the host• Produce disease symptoms• Transmission to others

The evolutionary success of a pathogen is measured by its ability to infect or colonize additional hosts, not in its

ability to kill.

All pathogens have developed ways to be transferred to others, and most have evolved to keep the host alive for a long enough to maximize their ability to pass

to other hosts.

Some pathogens such as HIV, which causes AIDS, do not

have to be transmitted efficiently.

They have figured out how to remain in a host long enough to maximize their chance to be passed to others

before the original host dies.

Pathogens such as Vibrio cholera (or Anthrax) don’t mind killing their host because in doing so they produce enough bacteria in a watery diarrhea (or spores on the savanna) to guaranty their transmission to a new host.

From the Perspective of the Host:

Host Defense Mechanisms

Major Host Defenses

Mechanical Barriersskinepithelial cellsmucusciliastomach acidproteases, etc

Phagocytesmacrophageskupffer cellsgranulocytes

Pattern Recognition Receptors

Toll-Like-Receptors (TLR)C-reactive proteinMannose-Binding Lectin

Complement

CytokinesInterferon a, b, gTNF-a, b,IL-1, IL-2, IL-4, IL-6, IL-17, etc.

AntibodyIgM, IgA, IgG, IgE

T-CellsCD4, CD8

NK-Cells

Relative Concentration ofSelected Immune Mediators

Mediator Conc./ml blood2µg/ml Specific Antibody 1013 molecules2ng/ml Specific Antibody 1010 molecules

PMN 5 x 106Mononuclear cells 1 x 106

CD4 T cells(antigen specific)

2 x 106(102-103)

CD8 T cells(antigen specific)

2 x 106(102-103)

Phagocytes (PMN & Macrophages)

Ingest and destroy microbesFind microbes by:

Chance encountersChemotaxis

bacterial breakdown products (N-formyl methionine)complement fragments (C5a)leukotriene from stimulated lymphocytes

Recognize microbes by molecules on their surfaceDeposited complement (C3b)Antibody FcRepeating structures on microbe surfaces (manose-binding lectin)

Protective Action of AntibodyAlone• blocks adherence• blocks toxin activity• blocks enzymatic activity

In conjunction with complement• aggregates and opsonizes toxins• aggregates and opsonizes microbes• blocks enzymatic activity• lyses host cells bearing bacterial or viral antigens

(containing bacteria or viruses)

• lyses Gram-negative bacteria

Membrane Attack Complex (MAC)

Complement’s role in opsonophagocytosis

C3 is activated• through the alternative pathway (factor B) by many microbial

surfaces. (Extracellular pathogens generally inhibit this pathway).

• through the classical pathway (C1q) by IgG and IgM antibody bound to pathogen surfaces.

• through a lectin pathway (mannose binding protein, L-ficolin, etc. )

C3 activation and surface deposition mediates• chemotaxis (C5a)• opsonization (C3b, iC3b)• lysis {membrane attack complex (C5b, C6, C7, C8, C9)}.

NK cellsLyse host cells identified by:

IgG (ADCC) -- kills cells infected with virus and bacteriatumor antigens -- tumor surveillance

Significant producer of g-interferonCell mediated immunity -- intracellular bacteria and viruses

Cytokines• provide communications between

immune cells• activate (or inactivate) immune cells • can stimulate protection against

microbial infections• can restore tissue to a non-

inflammatory state as an infection subsides

• mediate many of the inflammatory symptoms of infectious disease.

g interferon -- produced by immune T cells

a b interferons -- produced by infected cells

Recognition of Pathogens is facilitated by PRRs which recognize PAMPs

• PRR = Pattern Recognition Receptors– family of TLRs (toll-like receptors)– C-reactive protein– Mannose-binding lectin, etc.

• PAMP = Pathogen-Associated Molecular Patterns– Cell walls of bacteria– LPS of bacteria– Flagella of bacteria, – Microbial DNA and RNA, etc.

TLR4 is a PRR that recognizes LPS • Gram negative

bacteria all make LPS

• Mutant mice that lack TLR4 are much more susceptible to most gram negative bacteria.

• Yersinia pestis causes plague

• Y. pestis makes normal LPS in the flea (insect host)

• Y. pestis makes a variant LPS in mice to escape recognition by TLR4

• Mutants that make normal LPS in mice are totally avirulent.

Helper (CD4) T cells:

A. Activate (help) B cells to make antibody

B. Mediate cell-mediated immunity1. Enhance production of cytotoxic T cells2. Activate macrophages to kill intracellular

bacteria, and destroy infected host tissue.

Inflammation,

enhances microbial killing,

often at the expense of host tissue

Inflammation is mediated by,microbial products (LPS, cell wall, some toxins)

antibody and complementcell mediated immunity

Two types of pathogenic bacteria

• Extracellular -- replicate outside of cells and must therefore avoid being killed by phagocytes or complement.

• Intracellular -- replicate inside cells and must therefore avoid being killed inside phagocytes by the antibacterial properties of lysozomes.

Extracellular Bacteria

Host Resistance mediated by:AntibodyComplement

• opsonization of all bacteria• lysis of gram negative only

PhagocytesPattern Recognition Receptors

• TLRs, mannose binding proteins, etc.

Intracellular BacteriaResistance mediated by:

TH1 cellsMacrophagesCD8 T cells cellsNK cells (probably)

VirusesResistance mediated by:

antibody - to block extracellular spread

interferon - to block intracellular growth

NK and CD8 cells - to kill virus producing cells

Ten Minute Break

From the Perspective of the Pathogen

(Just looking for a warm damp place to live)

The special properties that allow pathogens to survive,

spread, and cause disease are called

Virulence Factors

Virulence Properties• Exploiting a particular niche• Adherence to host tissues• Partial (or complete) escape from host immunity

– Protects itself from Ab and C' attack– Invasion into cells & avoidance of degradation in lysozomes– Interference with host and/or adaptive immunity– Live in protected tissue sites

• Means of acquiring nutrients– Enzymes, siderophores, and transport machinery

• Mechanism for transmission to the next host

Finding a NicheStreptococcus

pneumoniaeAsymptomatic colonization of upper airways. Infects cavities (lung, ear, spinal fluid, eye, etc.)

Streptococcus mutans Adheres to glucan layer it applies to teeth

Helicobacter pylori Lives in mucus lining of the stomach

Listeria and Salmonella Survive in phagocytes

Salmonella (typhoid) Survive in bile and infect liver and spleen

E. coli Colonize gut, adhere to surface of bladder and ureter

Group B streptococci Asymptomatic colonization of gut, but can infect and kill newborn infants

Mycobacterium tuberculosis

Grows slowly to minimize elicitation of host immunity

HIV Grows in and kills CD4 T-cells, thus eliminating host anti-viral response

Adherence by many pathogens is important because,

1. It prevents them from being cleared by mucus flow.

2. It is necessary to subsequent to tissue and cellular invasion.

3. Presence on the mucosa is frequently critical for transmission.

Critical adhesion targets of some pathogens

Pathogen Host Tissue Attachment site on host cell

Influenzae virus Respiratory epithelium

Neuraminic acid

Mycoplasma pneumoniae

Respiratory epithelium

Neuraminic acid

Streptococcus mutans Tooth enamel Bacterial glucan

Neisseria gonorrhoeae Uretheral epithelium Carbohydrate on host cells

Escherichia coli Intestinal epithelium urinary tract epithelium

D-mannose

Malaria (Plasmodium vivax)

erythrocytes Duffy blood group antigen

Giardia lamblia Duodenum & jejunum

Mannose-6-phosphate

Bacterial Toxins

• Exotoxins -- secreted molecules that can kill, damage, or alter host cells – Diptheria toxin– cholera toxin– tetanus toxin, etc.

• Endotoxins -- Lipopolysaccharides (LPS) in the outer membrane of gram negative bacteria

Bacteria get iron via

• Siderophores (secreted molecules that bind iron ions) – Transport of siderophore+Fe+++

– Cytoplasmic degredation siderophore-Fe+++ to release Fe+++

• Transport of iron-binding transferrin, lactoferrin or heme.

• Growing intracellularly (where iron is more readily available)

Evasion of Phagocytosis

Structure Mechanism Pathogens Polysaccharide anti-complementary Streptococcus pneumoniae capsule antigenically unique Neisseria meningitidis Haemophilus influenzae M-protein anti-complementary Streptococcus pyogenes antigenically unique PspA & PspC anti-complementary Streptococcus pneumoniae Antigenic variation

create new antigens Neisseria gonorrhoeae

Pilin PII LOS

Evasion of Intracellular Killing

Pathogens Mechanism Mycobacterium

tuberculosis Glycolipid cell wall resists non-activated macrophages.

Slow growth fails to elicit strong Cell Mediated Immunity.

Listeria monocytogenes Listeriolysin - escape to cytoplasm

Actin tails - drive it into adjacent cells

Rickettsiae Escape to cytoplasm

Salmonella Prevents lysosome-phagosome fusion

Regulation of Virulence Factors in Response to Host Conditions

• Temperature• Carbon source• Concentration of Iron, Manganese, or

Calcium• Osmolarity• pH• Stress

Spread of Pathogens

Pathogens Mechanism Salmonella Spread in feces, survives in water

Cholera Induces tremendous volumes of diarrhea, Survives in water

Streptococcus mutans Saliva

Rhinoviruses Sneezing dripping noses

Syphilis, Chlamydia, Gonococci, HIV

Sex

Yersinia pestis, Lime disease,

St. Louis encephalitis virus & West Nile virus

Fleas & Rats Ticks & Deer Mosquitoes

Rabies Animal bites

Vaccines• Elicitation of Protective Immune

Response by Host.

- Antibody

- Cell Mediated Immunity

Vaccine ApproachesAntibody

Immunize with toxins (cholera, diphtheria, tetanus etc.)

Immunize with adhesions (flu)

Immunize with unique surface components(pneumococcus)

Cell Mediated Immunity

Intracellular bacteria and viruses

Polysaccharides are poorly immunogenic in children

less than two years of age.

Protein-Polysaccharide Conjugates

• To elicit a T-cell help dependent anti-polysaccharide response

Boostable response Higher titer response

• To permit immunization of young children• To better immunize the elderly

Polysaccharide-protein conjugate vaccines are available for

• group B Haemophilus influenzae

• Streptococcus pneumoniae (7 capsular types)

• Groups A and C Neisseria meningitidis.

Adjuvant• Most vaccines use alum (aluminum

hydroxide or aluminum phosphate) as an adjuvant) and are injected in muscle.

• Alum slows the release of antigen and causes inflammation that stimulates immune cells to make a response

Inclusion of cytokines and immune modulatorswith antigen

To enhance immune responses

To focus immune responses on

a. humoral

b. cell mediated

To focus immune responses on

a. TH1

b. TH2

• Mucosal Immunization– elicits systemic and mucosal immunity – avoids giving a shot– adjuvants for human use are still needed

• Mucosal Immunity can– block colonization and transmission (herd

immunity)– prevent invasion– block local action of toxins

Thanks for your attention

If you have questions,4-6595

or dbriles@uab.edu

Learn and understand concepts

Elements of Microbial Pathogenesis

1. Permissive Host / Virulent Pathogen

2. Pathogen Evades Host Defenses3. Pathogen Multiplies and Spreads

to others4. Disease/Symptoms/Inflammation

Extra Slides Follow.

• These slides may help with understanding but will not be explicitly covered by the exam questions.

Identify roles of immunity mediated byComplement and T-cells for two disease models in mice

Salmonella infection

(mouse typhoid)

Pneumococcal infection of the

blood

Eliminate complement C3 with Cobra Venom factor No Effect Increased

susceptibility

Eliminate T-cells with mutation

Increased susceptibility No effect

Pathogenesis involves complex in vivo events

Results obtained in vitro(outside the body)

must be verified by in vivo studies(inside the body)

Enterobactin (an example of a virulence factor)

• Enterobactin is an iron chelating siderophore released by enteric bacteria to bind iron

3+ and deliver it to the pathogen.

• Mutants unable to make enterobactin do not grow in the gut.

• Repair of mutation, or feeding enterobactin will permit growth.