The Human Microbiome and Pathogenicity

The Human Microbiome

Major components of the human microbiota, summarized across body sites:
- Gastrointestinal tract
- Oral cavity
- Vaginal mucosa
- Skin
Virus is higher
Humans as Habitats
- Warm, stable, lots of nutrients available, constant pH and osmotic pressure, etc.
- Our bodies are not uniform - each region or organ differs: skin, GI tract, respiratory tract, etc. provide different conditions
- Animals possess great defense mechanisms - the successful colonizers (and the successful pathogens) are those that can deal with these defenses
- Normal microflora doesn’t mean non-pathogenic; we often have low pathogens in low numbers in and on us

Humans as Habitats

Colonization (and infection frequently begin at mucous membranes
- These are found throughout the body.
- Consists of single or multiple layers of epithelial cells, tightly packed cells in direct contact with the external environment
- Bacteria may associate loosely or firmly
- Breaches in the mucosal barrier can result in infection (pathogenesis) by opportunistic pathogens
How did we get our indigenous microflora?
- Uterus environment totally sterile
- Initial colonization comes during breaking of fetal membranes and, especially, birth itself
- Environment in general is colonization source: mother, father
- Initial microflora of the gut depends on whether the infant is breastfed or not (bifidobacterium vs. others)
Effects of breastfeeding vs. bottle-feeding on microflora
- Large effect seen - breast fed infants develop primarily bifidobacterium populations, bottle-fed get a mixture of various species of coliforms
- Breastfed infants have a lower pH and less buffering capacity in large intestine - this disfavors enterobacteria and favors bifidobacteria
- Bifidobacterium seem to compete with potential pathogens like Clostridium difficile and some enterobacteria

Microbiota Diversity

Which has a more diverse microbiota?
- The large intestine of bottle-fed infants
Which is less acidic?
- The large intestine of bottle-fed infants

Microbiota of Specific Regions

Microorganisms are normally not found in the organs, blood, or lymph
Due to the extreme differences in these habitats, the microbiomes of the major areas of the body have different and distinct bacterial communities

Microbiota of the Human Skin

Skin surface is largely unfavorable habitat
- Exceptions are moisture areas: scalp, face, ears, underarms
- There are often many transient microbes (microorganisms that end up on your skin but they aren’t native there)
- Most resident skin microorganisms inhabit deeper layers of epidermis, sweat glands, and follicles
- Most of the residents are gram-positives, staphylococcus
- Commensal fungi form hyphae or exist as individual cells
- Skin mites live in and near hair follicles

Microorganisms Associated with Glands

Microorganisms are primarily associated with glands
1. Eccrine glands
  - Widely distributed
  - Main glands for perspiration; secrete a hypertonic saline solution with a variety of organic and inorganic substances
  - Relatively devoid of microorganism, probably due to salinity and low pH
2. Apocrine glands
  - Restricted to underarms, genitals, etc
  - Don’t develop before puberty
  - Apocrine sweat has higher pH than eccrine sweat
  - Population numbers can be high
3. Sebaceous glands - associated with hair follicles
  - Produce sebum, chief component of skin lipids
  - These lipids have antibacterial activity, esp. Against gram positives
As with the body as a whole, bacterial communities on different skin sites are themselves very different

Microbiota of the Human Mouth

The main ‘negatives’ for these bacteria:
- Salivary enzymes (lysozyme and lactoperoxidase)
- The constant need to re-attach
- As teeth erupt there are more anaerobes and bacteria adapted to living in crevices and on smooth services

Biofilm (plaque) formation

Begins as thin film of glycoproteins in saliva
Colonized quickly by individual Streptococcus cells, which grow to microcolonies
Extensive growth of these results in formation of a thick biofilm - further colonization can include filamentous forms, spirochetes, and various anaerobes
Dental plaque has structure
Plaque accumulates calcium salts to form tartar

Microbiota of the Human GI Tract

Where do you think the most bacteria are found?
- Large intestine
Stomach is the most acidic
Colon most pH balanced
What is the main cause of peptic ulcers?
- Infection

Stomach

pH of stomach is low, around 2 or less
Acts as a microbiological barrier
The infectious dose (the # of bacteria it takes to get sick)
Helicobacter pylori - cause of stomach ulcers (KNOW NAME)
The upper portions of the small intestine are acidic and resemble the stomach.
The lower portions have increasing numbers of bacteria from $10^5$ to $10^7$
The large intestine have enormous number of bacteria, > $10^{13}$
The major bacterial types in the GI tract
- Much more diverse bacteria in the large intestine
- There are different microenvironments in the large intestine
- Bacteroides - gram negative (main bacterium) - NEED TO KNOW
- Huge surface area in the large intestine

Probiotic therapy

Probiotic therapy: administering living bacterial cultures to confer a health benefit
- May be particularly useful for antibiotic-associated diarrhea: strain of Lactobacillus GG
Prebiotic - “feed” gut bacteria
Influence of gut microbiota on mouse behavior
- When mice with autistic-like behavior are fed human Bacteroides (#1 bacteria in our human gut) fragilis their condition is ameliorated

Microbiota of other body site

Urogenital tract
- Bladder usually sterile
- Urethra epithelium can be colonized by facultative Gram-neg. Rods like E. coli and especially Proteus that can become opportunistic urinary tract pathogens

Upper respiratory tract

Staphylococcus, streptococcus, diphtheroid bacilli, gram-neg. Cocci.
Also some pathogens

Lower respiratory tract

(in contrast) few microorganisms

Lecture 16: Pathogenicity and Infection I

Pathogen (or ‘true pathogen’) - can cause infection and disease in a healthy host
Opportunistic pathogen - only pathogenic to non-healthy individuals or when introduced into a normally sterile part of the body
Virulence - degree of pathogenicity of a parasite
Virulence factors - properties of the pathogen that allow it to successfully invade and cause disease in a host
E.coli is a - BOTH a true pathogen and opportunistic pathogen

Non-specific host defenses

Normal microbiota as defense
Anatomical defenses
Antibiotic displacement
- Opens up a niche - leaves for a space(niche) for new organism to slip into
Physical Barriers and Anatomical Defenses
- Skin, mucus, cilia, ear wax, sweat glands, stomach acid

Factors Relating to Susceptibility to Infectious Disease

Age - infants and elderly more susceptible. Why?
- Infants - Immune systems undeveloped, undeveloped normal flora
- Elderly - immune system is breaking down, immune response declines, anatomical changes
Stress - in rats: fatigue, exertion, poor diet, dehydration, drastic climatic changes increase incidence and severity of infections
Hormone imbalance plays important rule
Diet - famine and infectious disease correlated (e.g. cholera)

Events leading to infection

Starts with exposure
adherence (to skin or mucosa)
invasion - through epithelium (sometimes doesn’t happen)
Multiplication (grow in place and production of virulence factors and toxins)
Either Toxicity or Invasiness
- Toxicity - toxin effects are local or systemic
- Invasiveness - further growth at original and distant sites
- Together (toxicity and invasiness) cause tissue or systemic damage

STORCH

STORCH - Syphilis, Toxoplasmosis, Other disease (hepatitis B, HIV, chicken pox, and chlamydia), Rubella, Cytomegalovirus, and Herpes (KNOW)

Portals of entry

Portals of entry - characteristic route a microbe follows to enter the tissues of the body (eyes, ears, nose, mouth, placenta)
Exogenous agents - originate from source outside the body
Endogenous agents - already exist on or in the body (normal flora)

Penetration of host defenses

Tissue specificity (of the pathogen) is a serious barrier to the entry of most microorganisms
- Will be discussed in viruses, also true of other pathogenic microorganisms: usually, pathogens only infect specific tissues and cell types
- Some notable exceptions exist, e.g. Streptococcus pyogenes and Staphylococcus aureus
  - Pathogens must first become established at the site of infection. The habitat must be compatible with the microorganism
  - An infecting microorganism can’t adhere to all cells or hosts

Pathogens That Infect during Pregnancy

Attaching to the Host
- Adhesion - microbes gain a stable foothold at the portal of entry; dependent on binding between specific molecules on host and pathogen
- Fimbriae, flagella, Glycocalyx, cilia, spikes
- Capsules facilitate pathogen attachment
- Fimbriae (non-conjugative) pili, and even flagella can function by specifically binding to host cell surface glycoproteins
Bacterial adherence: receptors
- A bacterial pathogen attaches specifically to host tissues by way of complementary receptors on the bacterial and host surfaces

Virulence and Infectious Dose

Some pathogens are much more virulent than others
Infectious Dose (ID) - minimum number of microbes required for infection to proceed
Lack of sufficient ID typically means no infection
Microbes with small IDs have greater virulence
Some cells are pathogenic due to the toxins they produce but most need to actually invade and grow in host tissues in order to cause disease - growth causes the damage

Colonization, Growth, and Virulence

Colonization - multiplication of a microorganism after it has attached to host tissues or other surfaces
The initial inoculum of cells is rarely sufficient to cause disease; needs to grow
Must therefore find appropriate nutrients and environment. This not always as easy as it appears (e.g. iron)
Virulence Factor - any characteristic of a pathogen that enables it to establish itself and cause disease
These are often extracellular enzymes such as hemolysin, hyaluronidase, collagenase, and coagulase. The first 3 of these allow for spread ( an nutrition, to some extent), the 4th promotes localization and, probably, protection
The capsule can be a virulence factor in certain pathogens - True (capsules particularly colonizes lungs)
Exoenzymes - secreted enzymes - to digest food
Toxins - Activity of some enzyme virulence factors
Example of a virulence factor: urease production in H. pylori, the causal agent of stomach ulcers
- Doesn't want to grow in hot acids → moves deeper in gastric mucin
- H.pylori can raise the pH so it de-gels the mucin (causes mucin to liquefy), and the bacterium can swim right through

“Cards of Virulence”

Microbial card sets for four hypothetical microbes
Microbe 1 has the potential for being virulent in a mammalian host because it is the only one with the ability to grow at 37 degrees celsius

Surviving Host Defenses

Initial response of host defenses come from phagocytes
Antiphagocytic factors - used to avoid phagocytosis
Species of Staphylococcus and Streptococcus produce leukocidins, toxic to white blood cells
Slime layer or capsule - makes phagocytosis difficult
Ability to survive intracellular phagocytosis

Lecture 17: Pathogenicity and Infection II

Toxins
- Exotoxin - toxin secreted into tissue
- Target organs
  - Diphtheria toxin - extremely potent (one molecule will kill a cell). Disrupts protein synthesis. Caused by lysogenic bacteriophage (virus) found in Corynebacterium diphtheria
  - Tetanus and botulism toxins - casual organisms (Clostridium tetani and C. botulinum) don’t generally grow very much in infected tissues but instead release potent neurotoxins
  - Action of Tetanus Neurotoxin - causes irreversible muscle contraction (‘spastic paralysis’ or Lockjaw)
  - Action of Botulinum Neurotoxin - botulinum toxin, the most poisonous substance known, causes irreversible muscle relaxation (‘flaccid paralysis’)
Endotoxin - toxin released only upon cell death and lysis
- These are lipopolysaccharides and thus are found only in gram-negative organisms - most studied in Salmonella, E. coli, and Shigella
- General physiological effects
- Superantigens - excessive activation of the immune system, e.g. Toxic Shock Syndrome
- Membrane-disrupting toxins, e.g. streptolysin (S. pyogenes)
- Intracellular-targeting toxins (a.k.a) “A-B toxins”, e.g. cholera toxin

Types of Exotoxins

Enterotoxin - exotoxin that acts specifically on the small intestine
- Enterotoxins are found in S. aureus, enteropathogenic E. coli
- Most studied: Cholera toxin from Vibrio cholerae
- Action of cholera enterotoxin
  - Causes osmotic imbalance in small intestine
  - Inside of small intestine makes it salty in there - by osmosis water gets pulled out
  - Patterns of Infection
    - Mixed infection - multiple various microbes
    - Localized infection (boil)
    - Systemic infection (influenza)
    - Primary (urinary) infection → secondary (vaginal) infection

Nosocomial Infections

Nosocomial Infections (you pick it up at hospital)
- Diseases that are acquired or developed during a hospital stay
- From surgical procedures, equipment, personnel, and exposure to drug-resistant microorganisms
- 2 to 4 million cases/year in U.S. with approximately 90,000 deaths
- Preventing Nosocomial Infections (know each one/about)
- Type of isolation:
  - Enteric precautions - prevent spread of diarrheal diseases
  - Respiratory precautions - prevent spread of tuberculosis, measles, mumps
  - Drainage and Secretion Precautions - prevent spread of staphylococcal and streptococcal infections; gas gangrene; herpes, zoster
  - Strict isolation - mostly highly virulent or contagious microbes; includes tuberculosis, some types of pneumonia
  - Reverse isolation (also called protective isolation) - used to protect patients extremely immunocompromised by cancer therapy, surgery, genetic defects

Universal Blood and Body Fluid Precautions

Stringent measures to prevent the spread of nosocomial infections from patient to patient, from patient to worker, and from worker to patient - universal precautions
Based on assumption that all patient specimens could harbor infectious agents
The Immune System
Host Defense Mechanisms
- First line of defense: host physical and chemical barriers (skin, mucous, tears, etc.)
- Second line of defense: phagocytes and inflammation (non-specific)
- Third line of defense: specific immune reactions
- Summary of host defenses
  - First and second line of defense - nonspecific, inborn
  - Third line of defense - acquired, specific

Systems involved in immune defenses

Systems involved in immune defenses
1. Bloodstream
2. Lymphatic system
3. Reticuloendothelial (or mononuclear phagocyte) system - support system of connective tissue fibers and endothelium
4. Extracellular fluid

Communicating systems

Important blood cells involved in immune systems (KNOW)
- Stem cells - undifferentiated cells which give rise to all others. Found in bone marrow. Produces erythrocytes (RBCs), leukocytes (WBCs), and platelets
- Leukocytes - generally divided into 2 groups: granulocytes (polymorphonuclear leukocytes) and agranulocytes (mononuclear leukocytes)
- Examples of granulocytes: neutrophils (“microphage” - phagocytic), basophils, eosinophils
- Examples of agranulocytes: monocytes (phagocytic), lymphocytes

Agranulocytes

Lymphocytes
- T cells - derived from thymus. Important in cell-mediated immunity
- B cells - derived from bone marrow, primarily. Important in humoral (humor - fluid) Immunity
Monocytes - large, common, important in non-specific and specific immune functions. Non-circulatory (tissue-associated) monocytes are known as macrophages
- Humoral vs cell-mediated immunity:
  - Humoral type: antibody-mediated response
  - Antigen type: extracellular pathogens
  - B cells
  - Cell-mediated Type: t cell-mediated response
  - Antigen type: intracellular pathogens, cancer cells
  - T cells

Mast cells

Mast cells
- Similar function to basophils (inducing inflammatory response)
- Unlike basophils, mast cells found mainly in Natural Killer (NK) cells
  - Unlike other lymphocytes, are involved in non-specific immune response
  - NK cells recognize and destroy abnormal cells: cancer, infected cells

Types and functions of lymphocytes

Types and functions of lymphocytes
- B cell → activated → antibodies (Humoral immunity)
- T cell → activated → help, suppress, kill, hyper-sensitivity (cell-mediated immunity)
- The Non-specific immune response
  - Inflammation
  - Phagocytosis
  - Complement

Symptoms of inflammation

“Rubor” - redness
“Calor” - warmth
“Tumor” - swelling
“Dolor” - pain

Inflammation at the cellular level

Phagocytosis
- Carried out primarily by neutrophils and by monocytes/macrophages (collectively called ‘phagocytes’)
- Phagocytosis
  - After engulfment, phagocytes shift from aerobic to fermentative metabolism. Leads to drop in pH, which activates lysosomal enzymes
  - Phagocyte failure
    - Some pathogens can neutralize phagocyte products:
    - Staph. Aureus produces carotenoids which quench singlet oxygen
    - Mycobacterium sp. Can grow within macrophages using glycolipids in their cells walls to scavenge toxic oxygen species
    - Strep. Pyogenes and Staph. Aureus produce leukocidins that destroy phagocytes
    - The complement system

The complement system

“Complement” is an additional mechanism that is brought into play at several levels in cell killing
Is a cascade of blood proteins that results in membrane permeation and cell death

The Specific Immune Response: The Third (and Last) Line of Defense

Antigens: a macromolecule that reacts with components of the immune system
- Generally, bigger and more complex is better to illicit (immune) response
1. Whole cells or viruses
2. Proteins and other polypeptides
3. Lipo- and glycoproteins
4. Other complex polymers
Poor antigens: small, unattached molecules, simple molecules, large but repetitive

Antibodies

General structure: 4 protein chains in two pairs
What makes each antibody unique: variable region at the top
Some Antibody Functions:
- Bacterial cell “tagged” with Abs
- Opsonization
- Neutralization
- Agglutination - antibodies cross link bacterial cells and cause them to be in a clump (don't function anymore)
- Complement fixation (antibodies initiate so it happens more quickly and powerfully)
- Precipitation (Antibodies aggregate antigen molecules)
- Different kinds of antibodies (5 different kinds)
- Most common - IgG MAJOR
- Histocompatibility Complex (MHC) surface proteins
  - MHC I proteins are found on all nucleated cells
  - Necessary for cell recognition
  - MHC II proteins are found only on “antigen-presenting cells” like macrophages and B cells
  - Particularly important in T(helper) cell activation

The Specific Immune Response (The “Third Line of Defense”)

Engulfment, digestion, and presentation of antigens by a dendritic (phagocytic) cell
Lysosomes fuse with the phagosome and digest the bacterium
Immunodominant epitopes are associated with MHC II and presented on the cell surface
Overview of the Specific Immune Response: Lymphocyte Development
- Lymphocytes arise from the same stem cells but differentiate into two distinct cell types
- B cells mature in specialised bone marrow sites and T cells mature in the thymus gland
- Mature cells settle

Overview of the Specific Immune Response: Presentation and Activation

Monocytes (and sometimes B cells) come in contact with antigen (generally pieces of an invader)
Monocytes “present” antigen which activates specific T-helper cells
T-helper cells then activate specific B cells which produce antibodies

Overview of the Specific Immune Response: Initiation of Humoral Immunity

Activated B cells differentiate into Memory B cells and Plasma B Cells
Plasma cells go on to produce specific antibodies

Overview of the Specific Immune Response: Initiation of Cell-Mediated Immunity

“Naive” T cells are activated to form more T-helper, cytotoxic (‘killer’) T, and Memory T Cells
The Development of B Cells and T Cells

Generation of the amazing diversity of antigen receptors

Generation of the amazing diversity of antigen receptors: How the immune system can anticipate nearly any possible antigen
- As lymphocytes develop, genes involved in antigen binding are rearranged so each clone only produces a single receptor… forever
- Any clones that target “self” molecules are eliminated
- A specific antigen selects only the clone(s) that match it, initiating an immune response

Generation of Unique Clones with Unique Antibodies and Receptors

T-Cell Receptors
- Similar in function and structure to antibodies on B cells
- Variable (V) regions and Constant C regions
- Activation of T-helper cells by antigen presenting cells (APCs)
  - T-helper cell link to b-cell system?
  - Activation of B cells and Antibody Production
    - T helper cells has to find exact B-cell
    - Superantigens and Cytokine Storms
      - Macrophage present a superantigen that is not recognized by the TCR of the T cell, yet the superantigen still is able to bridge and bind the MHC II and TCR molecules

The Primary and Secondary Immune Responses

Primary response - a latent period with no measurable antibody occurs early on.
Second response - (more rapid) a latent period is lacking because other memory lymphocytes from the earlier response are immediately ready to react
Secretory IgA dimer - you get this if your mother breast feeds you (you get both IgG and IgA)

Lecture 18: Acellular Pathogens

Viruses
- Characteristics of Viruses
  - Somewhere between life and non-life
  - Can’t reproduce or metabolize independently
  - But can direct living cells to do so for them
  - All are “obligate intracellular parasites”
  - “Infectious particles” rather than “organisms”, “active”, or “inactive” rather than “alive” or “dead”
  - Possible exception: ‘Mimivirus’
    - 800 nanometer across (biggest virus in the world)
  - Virus Size
    - Viruses much smaller - nanometer range
    - General structure

Nucleocapsid

Naked virus
- Capsid composed of capsomeres
- Nucleic acid
Enveloped Virus
- All have a nucleocapsid composed of protein coat (capsid) and nucleic acid
- Some have phospholipid bilayer membrane
Viral Envelope
- Common in animal and plant viruses
- Envelope is required for infection
- Viral envelopes are actually portions of the infected host cell membrane (viruses do no form their own)
- Often replace host proteins with viral glycoproteins
- Coronavirus: an enveloped RNA virus that is the cause of COVID, Severe Acute Respiratory Syndrome (SARS)
- Nucleocapsid architecture
  - Two most common shapes: helical and icosahedral
  - Both are regular, geometric shapes determined by the structure and orientation of proteins in the nucleocapsid
  - Helical structure
  - Icosahedral
    - Composed of only one or two different proteins + nucleic acid
    - Adenoviruses - non-enveloped icosahedral viruses which are generally mild pathogens in humans, birds, etc. causing conjunctivitis (‘pink eye’)
    - Complex viruses
      - A number of types with somewhat more intricate structures
        One is typical bacteriophage, with icosahedral head, helical tail, fibers for attachment
        Phages - viruses that infect bacteria? Eat bacteria

Nucleic acid content

Viral genomes are very small
- 2000 bp (base pair) (enough to encode 3 or 4 proteins) to 200,000 bp Small compared to bacteria (~ $10^6$ bp) and human (~2 X $10^9$ )
- Characterized by (often) overlapping genes
- Viral genomes. The genomes of viruses can be either DNA or RNA (or some use both at different stages in their life cycle)
- Retroviruses (typically has RNA but can shift to DNA)

How viruses are classified

Based primarily on:
- Capsid structure: helical, icosahedral, or complex
- Enveloped or naked
- Type and structure of nucleic acid
  - DNA vs RNA
  - Single-stranded vs. double-stranded
  - Viral Infection - Overview
    1. Adsorption
    2. Entry (either of nucleocapsid or of nucleic acid only)
    3. Virion replication
      - Immediate viral replication (e.g. lytic phages) or
      - Integration of genome into host cell genome (e.g. lysogenic phase of temperate phages or integration of retroviruses) or
      - Latent infection (e.g. in herpes viruses)
    4. Exit from cell

Adsorption

Adsorption
Specific interaction of viral surface with cell surface
- HIV only infects cells (such as T cells) because it reacts with certain receptors

Entry

Injection of nucleic acid (e.g. T4 bacteriophage)
Entry of entire nucleocapsid (most common in animal and plant viruses)
Membrane fusion (only in enveloped viruses)

Viral Replication

Once inside, one of several things occurs, depending on the virus. However, the end result is intended to be new viruses (sooner or later)
Involves:
1. Replication of the nucleic acid
2. Synthesis of capsid proteins
3. Assembly of nucleocapsids

Exit

Cell lysis or
Budding
- Viral Life Cycles
  - Cultivating and Counting
    - Plates
      - Holes in the lawn - plaques
        Infected cells begin to grow but then are lysed, forming plaques in the lawn
        Plaques - where the bacteria is NOT because the phages kill bacteria
        One-step growth curve of virus replication
        Stagnant - eclipse period
        The latent period includes the eclipse and early maturation phases
        Lytic (or “virulent”) phage replication
        Attachment
        Penetration
        Biosynthesis
        Maturation
        Lysis
        Temperate phages have both lytic and lysogenic cycles (the lysogenic phase is when it ‘hides’ inside the genome)
The cell divides and prophage DNA is passed on to daughter cells → under stressful conditions, the prophage DNA is excised from the bacterial chromosome and enters the lytic cell
Latent infection
Some possible effects of animal virus infection of host cells
- Most animal viruses are lytic, and only a very few are known to cause cells to transform and become cancerous
- Viral Infection of plants
  - Virions rely on some form of surface damage to the plant, entering a plant cell through a wound or insect bite
  - Some bacteria are only pathogenic because of prophages? True

SARS- COV - 2

Viroids - strands of self-replicating RNA that can infect plants
Satellite RNAs - pathogenic RNAs that require a ‘helper’ virus to replicate
- Plant pathogenic type are called “virusoids”
- Prions
  - Infectious proteins
  - Cause of Transmissible Spongiform Encephalopathy
    - Human: creutzfeld-Jakob disease, Kuru
    - Animal: mad cow disease, scrapies (sheeps and goats)

How prions functions

Infectious prions convert normal prions into more infectious prions by folding them into beta-pleated sheets