I. THE INNATE IMMUNE SYSTEM

F. PHAGOCYTOSIS

2. The Steps Involved in Phagocytosis

The overall purpose of this Learning Object is:
1) to learn the steps involved in the phagocytosis and destruction of microbes;
2) to introduce several mechanisms microbes use to resist phagocytosis and destruction; and
3) to give several examples of microbes that utilize such mechanisms.

LEARNING OBJECTIVES FOR THIS SECTION

Innate immunity refers to antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to an antigen (def). This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.

Unlike adaptive immunity, innate immunity does not recognize every possible antigen. Instead, it is designed to recognize molecules shared by groups of related microbes that are essential for the survival of those organisms and are not found associated with mammalian cells. These unique microbial molecules are called pathogen-associated molecular patterns or PAMPS and include LPS from the gram-negative cell wall, peptidoglycan and lipotechoic acids from the gram-positive cell wall, the sugar mannose (a terminal sugar common in microbial glycolipids and glycoproteins but rare in those of humans), bacterial and viral unmethylated CpG DNA, bacterial flagellin, the amino acid N-formylmethionine found in bacterial proteins, double-stranded and single-stranded RNA from viruses, and glucans from fungal cell walls. In addition, unique molecules displayed on stressed, injured, infected, or transformed human cells also act as PAMPS. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometimes referred to as microbe-associated molecular patterns or MAMPs.)

Most body defense cells have pattern-recognition receptors for these common PAMPSand so there is an immediate response against the invading microorganism. Pathogen-associated molecular patterns can also be recognized by a series of soluble pattern-recognition receptors in the blood that function as opsonins and initiate the complement pathways. In all, the innate immune system is thought to recognize approximately 103 of these microbial molecular patterns.

The innate immune responses do not improve with repeated exposure to a given infection and involve the following:

Examples of innate immunity include anatomical barriers, mechanical removal, bacterial antagonism, pattern-recognition receptors, antigen-nonspecific defense chemicals, the complement pathways, phagocytosis, inflammation, and fever.

We will now take a closer look at the process of phagocytosis.

2. The Steps Involved in Phagocytosis

There are a number of distinct steps involved in phagocytosis:

a. Activation of the Phagocyte

Resting phagocytes are activated by inflammatory mediators such as bacterial products, complement proteins (def), inflammatory cytokines (def), and prostaglandins (def). As a result, the circulating phagocytes produce surface glycoprotein receptors that increase their ability to adhere to the inner surface of capillary walls, enabling them to squeeze out of the capillary and be attracted to the site of infection.

In addition, they produce endocytic pattern-recognition receptors (def) that recognize and bind to pathogen-associated molecular patterns or PAMPs (def) - components of common microbial molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, and mannose that are not found in human cells - to attach the microbe to the phagocyte for what is called unenhanced attachment (discussed below).

They also exhibit increased metabolic and microbicidal activity by increasing their production of ATPs, lysosomal enzymes, lethal oxidants, etc.

 

b. Chemotaxis of Phagocytes (for wandering macrophages, neutrophils, and eosinophils)

Chemotaxis is the movement of phagocytes toward an increasing concentration of some attractant such as bacterial factors (bacterial proteins, capsules, LPS, peptidoglycan, teichoic acids, etc.), complement proteins (C5a), chemokines (chemotactic cytokines such as interleukin-8 secreted by various cells), fibrin split products, kinins, and phospholipids released by injured host cells.


Movie showing chemotaxis by neutrophil. Chemotaxis of Neutrophils. © From Intimate Strangers: Unseen Life on Earth. Created by Mondo Media. Peter Baker, Executive Producer. Licensed for use, ASM MicrobeLibrary.


 

Some microbes, such as the influenza A viruses , Mycobacterium tuberculosis (inf), blood invasive strains of Neisseria gonorrhoeae (inf), and Bordetella pertussis (inf) have been shown to block chemotaxis.

 

c. Attachment of the Phagocyte to the Microbe or Cell

Attachment of microorganisms is necessary for ingestion. Attachment may be unenhanced or enhanced.

1. Unenhanced attachment
Unenhanced attachment is the innate recognition of pathogen-associated molecular patterns or PAMPs (def) - components of common molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, mannans, and glucans common in microbial cell walls but not found on human cells - by means of endocytic pattern-recognition receptors (def), such as scavenger receptors and mannose receptors, on the surface of the phagocytes (see Fig. 1).

 

2. Enhanced attachment
Enhanced attachment is the attachment of microbes to phagocytes by way of an antibody molecule (def) called IgG, the complement (def) proteins C3b and C4b produced during the complement pathways (see Fig. 2), and acute phase proteins (def) such as mannose-binding lectin (MBL) and C-reactive protein (CRP). Molecules such as IgG, C3b, and MLB that promote enhanced attachment are called opsonins (def) and the process is also known as opsonization (def). Enhanced attachment is much more specific and efficient than unenhanced.

3. Extracellular trapping with NETs (def)
In response to certain pathogen associated molecular patterns such as LPS, and certain cytokines such as IL-8, neutrophils release DNA and antimicrobial granular proteins. These neutrophil extracellular traps (NETs) bind to bacteria, prevent them from spreading, and kill them with antimicrobial proteins (see Fig. 15 and Fig. 16).

 

Some microorganisms are more resistant to phagocytic attachment.

  • Some capsules prevent the formation of C3 convertase, an early enzyme in the complement pathways. Without this enzyme, the opsonins C3b and C4b, as well as the other beneficial proteins are not produced.

  • Other capsules, rich in sialic acid, a common component of host cell glycoprotein, have an affinity for serum protein H, a complement regulatory protein that leads to the degradation of the opsonin C3b by factor I and the formation of C3 convertase. (Serum protein H is what normally leads to the degradation of any C3b that binds to host glycoproteins so that we don't stick our own phagocytes to our own cells with C3b.)

  • Some capsules simply cover the C3b that does bind to the bacterial surface and prevent the C3b receptor on phagocytes from making contact with the C3b (see Fig. 3). This is seen with the capsule of Streptococcus pneumoniae.

  • Neisseria meningitidis (inf) has a capsule composed of sialic acid while Streptococcus pyogenes (group A beta streptococci) (inf) has a capsule made of hyaluronic acid. Both of these polysaccharides closely resemble carbohydrates found in human tissue polysaccharides and because they are not recognized as foreign by the lymphocytes that carry out the immune responses, antibodies are not made against these capsules. Likewise, some bacteria are able to coat themselves with host proteins such as fibronectin, lactferrin, or transferrin and in this way avoid antibodies.

The body's immune defenses, however, can eventually get around the capsule by producing opsonizing antibodies (IgG) against the capsule. The antibody then sticks the capsule to the phagocyte. In vaccines against pneumococccal pneumonia (inf) and Haemophilus influenzae type b (inf), it is capsular polysaccharide that is given as the antigen (def) in order to stimulate the body to make opsonizing antibodies against the encapsulated bacterium.

  • An outer membrane molecule of Neisseria gonorrhoeae (inf) called Protein II and the M-protein of Streptococcus pyogenes (inf) allow these bacteria to be more resistant to phagocytic engulfment. The M-protein of S. pyogenes (inf), for example, binds factor H of the complement pathway and this results in the degradation of the opsonin C3b by factor I and the formation of C3 convertase. S. pyogenes also produces a protease that cleaves the complement protein C5a.

  • Staphylococcus aureus (inf) produces protein A while Streptococcus pyogenes (inf) produces protein G. Both of these proteins bind to the Fc portion of antibodies (see Fig. 4) and in this way the bacteria become coated with antibodies in a way that does not result in opsonization (see Fig. 5).

 

d. Ingestion of the Microbe or Cell by the Phagocyte

Following attachment, polymerization and then depolymerization of actin filaments (def) send pseudopods out to engulf the microbe (see Fig. 6) and place it in an endocytic vesicle called a phagosome (def) (see Fig. 7).

Scanning electron micrographs of a macrophage with pseudopods and a macrophage phagocytozing E. coli on a blood vessel; courtesy of Dennis Kunkel's Microscopy.


 

Some microorganisms are more resistant to phagocytic ingestion

  • Pathogenic Yersinia, such as the one that causes plague, contact phagocytes and, by means of a type III secretion system, deliver proteins which depolymerize the actin microfilaments (def) needed for phagocytic engulfment into the phagocytes (see Fig. 8). Another Yersinia protein degrades C3b and C5a.

 

e. Destruction of the Microbe or Cell

Phagocytes contain membranous sacs called lysosomes (def) produced by the Golgi apparatus that contain various digestive enzymes, microbicidal chemicals, and toxic oxygen radicals. The lysosomes fuse with the phagosomes containing the ingested microbes and the microbes are destroyed (see Fig. 9).

To view an electron micrograph of a phagolysosome, see the Web page for the University of Illinois College of Medicine.

Some bacteria are more resistant to phagocytic destruction once engulfed.
  • Some bacteria, such as Legionella pneumophilia (inf) and Mycobacterium species (inf), cause the phagocytic cell to place them into an endocytic vaculole via a pathway that decreases their exposure to toxic oxygen compounds.

  • Some bacteria, such as Salmonella (inf), are more resitant to toxic forms of oxygen and to defensins (toxic peptides that kill bacteria).

  • Some bacteria, such as Shigella flexneri (inf) and the spotted fever Rickettsia (inf), escape from the phagosome into the cytoplasm prior to the phagosome fusing with a lysosome (see Fig. 10).
  • Some bacteria, such as species of Salmonella (inf), Mycobacterium (inf), Legionella (inf), and Chlamydia (inf), block the vesicular transport machinery that enables the phagosome to fuse with the lysosome.
  • Some bacteria, such as pathogenic Mycobacterium (inf) and Legionella pneumophilia (inf), prevent the acidification of the phagosome which is needed for effective killing of microbes by lysosomal enzymes. (Normally after the phagosome forms, the contents become acidified because the lysosomal enzymes used for killing function much more effectively at an acidic pH.)

  • Some bacteria are able to kill phagocytes. Bacteria such as Staphylococcus aureus (inf) and Streptococcus pyogenes (inf) produce the exotoxin leukocidin which damages either the cytoplasmic membrane of the phagocyte or the membranes of the lysosomes (resulting in the phagocyte being killed by its own enzymes). On the other hand, bacteria, such as Shigella (inf) and Salmonella (inf), induce macrophage apoptosis, a programmed cell death.

If the the infection site contains very large numbers of microorganisms and high levels of inflammatory cytokines and chemokines are being produced in response to PAMPs , the phagocyte will empty the contents of its lysosomes by a process called degranulation in order to kill the microorganisms or cell extracellularly (inf).

These released lysosomal contents, however, also kill surrounding host cells and tissue. Most tissue destruction associated with infections is a result of this process (see Fig. 11). This is discussed later in Unit 4 under chronic inflammation.

The phagocyte will also empty the contents of its lysosomes for extracellular killing if the cell to which the phagocyte adheres is too large to be engulfed (see Fig. 12 and Fig. 13).

There are 2 killing systems in neutrophils and macrophages: the oxygen-dependent system and the oxygen-independent system.

1. the oxygen-dependent system: production of reactive oxygen species (ROS)

The cytoplasmic membrane of phagocytes contains the enzyme oxidase which converts oxygen into superoxide anion (O2-). This can combine with water by way of the enzyme dismutase to form hydrogen peroxide (H2O2) and hydroxyl (OH) radicals.

In the case of neutrophils, but not macrophages, the hydrogen peroxide can then combine with chloride (Cl2-) ions by the action of the enzyme myeloperoxidase (MPO) to form hypochlorous acid (HOCL), and singlet oxygen.

In macrophages, nitric oxide (NO) can combine with hydrogen peroxide to form peroxynitrite radicals. (In addition to ROS and NO, macrophages secrete inflammatory cytokines such as TNF-alpha, IL-1, IL-8, and IL-12 to promote an inflammatory response.)

These compounds are very microbicidal because they are powerful oxidizing agents which oxidize (def) most of the chemical groups found in proteins, enzymes, carbohydrates, DNA, and lipids. Lipid oxidation can break down cytoplasmic membranes. Collectively, these oxidizing free radicals are called reactive oxygen species (ROS).

Oxidase also acts as an electron pump that brings protons (H+) into the phagosome. This lowers the pH within the phagosome so that when lysosomes fuse with the phagosome, the pH is correct for the acid hydrolases, like elastase, to effectively break down cellular proteins.

In addition to phagocytes using this oxygen-dependant system to kill microbes intracellularly, neutrophils also routinely release these oxidizing agents, as well as acid hydrolases, for the purpose of killing microbes extracellularly. These agents, however, also wind up killing the neutrophils themselves as well as some surrounding body cells and tissues as mentioned above.

To view a QuickTime video showing the oxidative burst of leukocytes, see "Ouch!" under the CELL'S ALIVE web page. For an article on toxic oxygen radicals, see "What the Heck Do You Mean - Oxygen is Harmful" on John Brown's Bugs in the News web page.

2. oxygen-independent system

Some lysosomes contain defensins (def)), cationic (def) peptides that alter cytoplasmic membranes; lysozyme (def), an enzyme that breaks down peptidoglycan, lactoferrin (def); a protein that deprives bacteria of needed iron; cathepsin G (def), a protease that causes damage to microbial membranes; elastase (def); a protease that kills many types of bacteria; bactericidal permeability increasing protein (BPI (def)), proteins used by neutrophils to kill certain bacteria by damaging their membranes; collagenase (def);and various other digestive enzymes that exhibit antimicrobial activity by breaking down proteins, RNA, phosphate compounds, lipids, and carbohydrates.

 

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