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Infectious Diseases in Clinical Practice:
doi: 10.1097/01.idc.0000240862.74564.57
Immunology/Microbiology for ID

The Bacterial Cell Wall: The Armor, Artillery, and Achilles Heel

Bower, Sam*; Rosenthal, Ken S. PhD†

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*Geneva College, Beaver Falls, PA and †Department of Microbiology, Immunology and Biochemistry, Northeastern Ohio Universities College of Medicine, Rootstown, OH.

Ken S. Rosenthal has research support from the NIAID, National Institutes of Health.

Address correspondence and reprint requests to Ken S. Rosenthal, PhD, Northeastern Ohio Universities College of Medicine, 4209 SR 44, Rootstown, OH 44272. E-mail: ksr@neoucom.edu.

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Abstract

Abstract: The bacterial cell wall, like the outer shell of a submarine or a spaceship, is the armor of the bacteria which contains and protects its cytoplasmic contents. The cell wall also contains attachment, invasion, and toxin-secretory weapon systems (artillery). The importance of the cell wall makes it an Achilles heel, and disruption of the cell wall kills the bacteria. This review provides an overview of the structure of the bacterial cell wall, its components and their role as virulence factors, and the vulnerability of the cell wall as a target for antimicrobials and host innate and immune defenses.

When I think of the bacterial cell wall, I think of the outer shell of a submarine or a spaceship. Like a submarine or spaceship, bacteria must be self-sufficient, as it floats through its liquid environment. Each of them is surrounded by a shell that must protect and contain its contents. The shell must also be able to allow selective entry of foodstuffs and beneficial materials while restricting entry of harmful things. These shells must withstand great pressures, and a failure in their structural integrity will cause them to burst. The outer shell provides an ability to attach and dock with structures and facilitate invasion. Weapon systems are delivered from and through the shell (eg, torpedoes). The outside shell is the first part of each of these entities to be seen and can set off alarms which can activate defenses to promote its destruction. This attack may also lead to peripheral damage to the "defenders." For these reasons, the bacterial cell wall acts as the armor for the cell and provides virulence weapons (artillery) to the cells but also provides vulnerability (Achilles heel) if it is not synthesized properly or its structure is disrupted. In this review, the structure, synthesis, virulence properties, and targets for antibacterial action of the cell wall will be discussed, and new concepts regarding the bacterial cytoskeleton and invasive mechanisms will be highlighted.

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The Armor

The cell wall of a bacterium is its skin and its armor. The basic bacterial cell wall consists of a plasma membrane similar to eukaryotic membranes and a meshlike peptidoglycan. Depending upon the type of bacteria, an outer membrane or a waxy layer may be added. These structures surround the cell as a container for the enzymatic and ribosomal machines, the chromosome and the proteins, metabolites, and ionic buffer solutions within the cytoplasm that power and promote the bacteria.

The plasma membrane, like the inner walls of a spaceship or submarine, is lined and penetrated with proteinaceous devices for metabolism, portals for internalization and secretion of biomolecules and ions, and molecular dynamos for producing energy. For example, it is the site where the F1 adenosine triphosphase (ATPase) converts chemoelectrical energy into ATP for the cell. Contrary to what many textbooks say, a relatively new finding demonstrates that a cytoskeleton on the inside of the plasma membrane contributes to the structural integrity (armor and shape) of the bacteria.1 The FtsZ, the "z protein," was discovered first, its structure is like that of eukaryotic tubulin, and it has a function in cell division.2 MreB and Mbl are proteins similar to the eukaryotic protein actin and play important roles in maintaining the shape of the cell. Related proteins have been discovered in many different types of bacteria. Proteins which block the function of the FtsZ protein also block bacterial cell division.3

The cytoskeleton of bacteria is supplemented by an exoskeleton to allow the bacteria to live in hypotonic solutions as dilute as distilled water and withstand osmotic pressures across the bacterial membrane equivalent to 25 atmospheres, similar to the pressures on a submarine hull or the shell of a spaceship. The meshlike exoskeleton of the peptidoglycan surrounds the plasma membrane and provides the strength to prevent cell lysis under these conditions (Fig. 1). The peptidoglycan consists of a polysaccharide backbone made up of repeating disaccharides of N-acetyl glucosamine (G) and N-acetyl muramic acid (M) which are cross-linked by peptide chains attached to the N-acetyl muramic acid. For the gram-positive bacteria, the peptidoglycan layer is very thick, and this serves as a protective mesh against the elements. The cell wall closest to the plasma membrane is tightly cross-linked, whereas the more outer layers are looser and subject to degradation and turnover by bacterial autolysins, such as lysozyme.

Figure 1
Figure 1
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The gram-negative bacteria have a single layer of peptidoglycan which is adjacent to and surrounded by an outer membrane. The periplasmic space separates the plasma membrane from the peptidoglycan/outer membrane shell. The periplasmic space contains proteins that facilitate the transport of molecules into the cell and other proteins waiting to be secreted from the cell. The outer membrane has a unique composition and structure which provides the strength to compensate for the thinness of the peptidoglycan. The acid-fast bacteria are gram-positive bacteria which supplement the peptidoglycan layer with a wax like coat.

Mother nature helps all of us microbiologists to distinguish the different bacterial membrane structures because they stain differently with the Gram and acid-fast stains. The thick peptidoglycan layers of gram-positive bacteria trap the precipitated dye (crystal violet) from the Gram stain to stain them purple (P = Purple = Positive). The single layer of peptidoglycan for gram-negative bacteria is insufficient to trap the Gram stain, it is washed away, and these bacteria must be counter stained with safranin (red color). The acid-fast bacteria stain gram positive but will also stain red with the acid stain.

For gram-positive bacteria, teichoic acid and lipoteichoic acid molecules weave through the thick peptidoglycan. Teichoic acids are polymers of chemically modified alcohols (eg, glycerol phosphate or ribitol). Teichoic acids are chemically attached to the peptidoglycan, and lipoteichoic acids are anchored by a fatty acid in the plasma membrane. Teichoic acids help protect the surface of the bacteria from autolysins and function as surface antigens for some gram-positive bacteria. Some of the teichoic acids of Streptococcus pneumoniae are known as the Forssman antigens and help to serologically group the streptococcus bacteria.

For gram-negative bacteria, the outer membrane is essential for the viability of the bacteria. The inside leaflet of the outer membrane consists of phospholipids and proteins, such as most membranes. The outer leaflet of the membrane is very different from other membranes and consists of protein and lipopolysaccharides (LPS). Lipopolysaccharide consists of 3 parts: the lipid A, the core, and the long, repeating O antigen (Fig. 1C). The lipid A has an unusual lipid structure with fatty acids attached to a disaccharide backbone. In addition, individual lipid A molecules can be connected through phosphate bonds to form tight extracellular membrane panels. The lipid A structure is so unusual that it is one of the strongest activators of human innate protective responses as endotoxin (discussed later). The core unit is a branched polysaccharide of 9 to 12 sugars. Lipid A and most of the core unit are essential for LPS structure and bacterial viability. Lipopolysaccharide molecules are connected by divalent cation bridges between phosphates on the lipid A and within the core region to create a tight, impervious structure. The O antigen portion of LPS consists of 50 to 100 repetitive units of 4 to 7 sugars. Differences in the O antigen portion distinguish different strains of bacteria, for example, enterohemorrhagic Escherichia coli expresses the O antigen defined as O157:H7. The O antigen provides a slimy hydrophilic outer layer that hides the cell surface from the complement system and its membrane attack complex.

The linkage of the individual LPS molecules through covalent phosphate bonds and divalent cation (Ca+2 and Mg+2) cross-linking bridges creates an interlocked outer wall for the bacteria. The tight outer layer provides a structural barrier to help the bacteria withstand osmotic shock as well as a barrier to toxic enzymes and antimicrobial compounds. Displacement of the divalent cations by positively charged peptides (antibiotics) or removal of the divalent cation with EDTA, a chelator, will disrupt the membrane (see Achilles Heel).

Neisseria species have a version of LPS which lacks the O antigen and is called lipooligosaccharide (LOS). The LOS is shed more readily than the LPS, and this contributes to the virulence and disease presentation of these organisms.

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Camouflage

Many bacteria have an additional layer of soft armor/camouflage, a capsule, on top of their cell wall or outer membrane consisting of proteins or carbohydrates which protect or hide the bacteria from recognition by the host innate and/or immune responses. These "cloaking devices" (ala Star Trek) are major weapons for the bacteria. The capsule is made up of loosely attached polysaccharide which acts like a slimy, tear-away shirt that is difficult to antigenically recognize and is antiphagocytic because it is difficult to grab by leukocytes. The capsule of Streptococcus pyogenes is composed of hyaluronic acid, the same polymer found in human connective tissue, and this antigenic disguise prevents recognition of the streptococci by phagocytes or the immune system. For the meningitis-causing S. pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, the capsule prolongs the presence of the bacteria in the bloodstream long enough to enter the meninges and also evade phagocytic control. For Streptococcus mutans and pseudomonas species, the capsular polysaccharide can build up into a biofilm to protect the colony from host defenses, detergents, and other toxic molecules and promote adherence to surfaces.

Specific proteins can also provide camouflage. The M protein of group A streptococcus is a major virulence factor providing protection from complement and phagocytosis by binding to serum factor H, a complement control protein.4-6 The M protein is a fibrous protein whose C terminus is attached to the peptidoglycan layer or embedded in the plasma membrane and penetrates through the cell wall. The protein contains an N-terminal hypervariable region with more than 80 different known serotypes. Some M proteins mimic the antigenicity of human cardiac muscle which may hide them from the immune response but also contributes to the development of rheumatic fever. Group B streptococcus bacteria, E. coli, and some neisseria use a surface coat of sialic acid to promote factor H binding and protection from complement. Streptococcus aureus can be camouflaged by coating itself with antibody molecules bound to its protein A. Protein A is attached to the peptidoglycan and binds the Fc portion of immunoglobulin G which prevents the antibody from interacting with complement or Fc receptors on white blood cells. Streptococcus aureus and S. pyogenes can also coat themselves with coagulation factors such as fibrinogen, factors V, XI, and XII, and H-kininogen and convert fibrinogen to fibrin to wall off the infection site from host defenses.

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Portholes in the Shell

As with a submarine or spaceship, there are specially designed portholes and mechanisms for bringing in material such as food and water and mechanisms for releasing waste material, or shooting off weapons. Special mechanisms are necessary to selectively secrete proteins from the cell without compromising the bacterial hull and causing leakage of small molecules and ions. Transport proteins and channels in the plasma membrane facilitate or carry material into and out of the bacteria. Specific unfolded proteins which have a defined peptide signal sequence, a ticket, are transported by the (sec) secretory system. The "peptide ticket" can be clipped off upon release. This is the same type of system that transports proteins into the endoplasmic reticulum of eukaryotes for glycosylation. The tat system can transport native proteins across the plasma membrane.7

For the gram-negative bacteria, the outer membrane represents an additional barrier to the uptake and delivery of molecules. The outer membrane is traversed by porin channels. The porin channels are made up of 3 individual porin subunits which provide selective entry for small, hydrophilic molecules while restricting larger and hydrophobic molecules such as bile salts, toxic molecules, lysozyme, and many antibacterial drugs. Secretion across the outer membrane also requires special mechanisms. Gram-negative bacteria have specialized molecular portals in their outer membrane hull to secrete molecules; many of these molecules are weapons, such as toxins. These portals have been grouped and characterized as types I to V secretory systems and will be discussed as weapons in the Artillery section.

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Antennae

The bacterial surface has sensors which can detect and respond to changes in temperature, osmolarity, light, oxygen, nutrients, small molecular signals, etc. Upon sensing the change in these parameters, a molecular signal is produced, sent to the genome of the cell to up- or down-regulate the expression of effector proteins. For example, many enteric bacteria make a large switch in the expression of metabolic enzymes when they sense a change in temperature upon "transition" from the bowel of a cow to a pile on the field. Researchers recently reported that salmonella and enterohemorrhagic E. coli can detect when it reaches the intestine of the body by sensing hormones such as epinephrine or even signals from fellow intestinal bacteria.8 Upon sensing that it is in the intestine, the E. coli activates its machinery, virulence factors, to colonize the intestine.

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Mooring Lines and Propellers

Docking mechanisms are provided by pili, which interact with other bacteria for conjugation (the closest thing to sex that a bacteria can experience) and, as fimbriae, with host cell surfaces. Pili are made of individual pilin proteins which form a hollow fiber with sticky adhesion proteins at the end. The flagellum is the propeller of the bacteria, and this outboard motor invented by mother nature could have been designed by Evinrude or Mercury. It consists of 3 basic parts, a filament, a hook, and a basal body. The filament is a hollow tube composed of flagellin which extends away from the cell surface. A bend in the filament just above the outer membrane, the hook, allows the flagella to point away from the cell surface. The basal body consists of a shaft and a series of protein rings that anchor the flagella to the cell membranes. Gram-positive organisms have 2 anchoring rings, 1 in the cell wall, and 1 in the cell membrane, whereas gram-negative organisms have 4 rings, to anchor in the cell membrane, cell wall, and the outer membrane. In addition to anchoring the flagella, the basal body also functions as an electrical motor using the membrane potential to power the rotation of the flagella to propel the bacteria. There may be only one or many flagella on a bacteria. The bacteria use the flagella to swim toward food and away from toxic molecules (chemotaxis). Vibrio cholerae swims (laterally) into the intestinal mucosa to avoid being flushed out by peristaltic action.9 Helicobacter pylori use flagella to penetrate into the gastric mucous layer which provides protection from the acidic environment.10

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Synthesis and Repair of the Armor

As for a space station, bacteria must be able to grow and expand their outer walls, but expansion requires building onto the structures that are outside of the cell. The trick that space stations, submerged submarines (or submerged city), and bacteria use for expansion is to prepare prefabricated units that fit together on the outside. Bacteria prepare subunits of cell wall components in the cytoplasm. The building blocks are attached and assembled on a molecular conveyor belt/assembly line (bactoprenol) which then flips the components to the outside of the cell. Each of the prefab units is activated for assembly onto the outer surface structure. Enzymatic machines, like a deep-sea diver or astronaut, provide the tools for the final assembly. Peptidoglycan, lipopolysaccharide, and teichoic acid are synthesized by this approach. For peptidoglycan, uridine-diphosphate-N-acetyl muramic acid acquires a pentapeptide chain in the cytoplasm and attaches to the bactoprenol molecular conveyor belt which is associated with the cytoplasmic membrane. The N-acetyl glucosamine (G) is then attached (for staphylococci, a pentaglycine chain is attached to the peptide portion) and then, the unit is translocated to the outer surface of the cell. The pyrophosphate linkage between M and the bactoprenol provides the chemical energy to power the attachment of the G-M unit to a polysaccharide chain of the peptidoglycan. The cross-linking of the peptidoglycan chains is energized by the peptide bond between the last 2 amino acids of the peptide chain, both of which are D-alanines. The energy of this bond is used to make a peptide bond between the free amine of lysine (or other attached amino acid in the third position of the peptide chain) to cross-link the peptidoglycan. Cross-linking is essential for continued synthesis of the peptidoglycan and for its strength, and the enzymes involved in cross-linking are targets for β-lactam and vancomycin antimicrobials.

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Artillery (Weapons-Virulence Mechanisms)

The weapons of a submarine or spaceship are displayed or must pass through the outer shell on their way to engage the enemy. Bacteria decorate their cell wall with many of their weapons and provide channels and devices to shoot off other weapons. These cell wall weapons are used to establish a toehold within our bodies (adherence), to invade our cells and tissues (invasion) and as suicide bombs to activate tissue damaging (inflammation) and systemic (sepsis) host protective responses to the infection. Some of the host responses to the cell wall components are so fanatical that they become responsible for the symptoms of microbial disease.11

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Tying Up to the Pier and Establishing a Toehold

Bacteria establish a toehold in the body by attaching themselves to cell surfaces using special adherence structures (Table 1).12 Adherence prevents them from being washed or carried out of their niche within the body. For enteric bacteria, this niche would be the kidneys, bladder, and gastrointestinal (GI) tract. Similarly, respiratory bacteria adhere to tissue surfaces to prevent themselves from being "blown" or swept away by the flow of mucous. The mooring lines are fimbriae which have adhesive structures, termed adhesins at their tips. The fimbriae are specialized pili, fiberlike structures which stick out from the surface of the bacteria. Most fimbriae are capped by lectins which allow the bacteria to bind selectively to specific surface carbohydrates. Lectins bind to specific sugars. The type 1 and P fimbriae of E. coli bind the bacteria to mannose and the P blood group glycolipid, respectively, on erythrocytes and uroepithelial cells to facilitate bladder and urinary tract infections. The pili of Neisseria gonorrhoeae are important virulence factors and promote binding to mucoepithelial cells.13 Other bacteria use alternative cell wall components for adherence. Streptococcus pyogenes uses its teichoic acid and F protein, and Treponema pallidum (syphilis) uses its P1, P2, and P3 proteins to adhere to fibronectin found on the surface of mucoepithelial cells.14,15 Lectins associated with the outer bacterial surface can also promote bacterial binding to mannose, fucose, N-acetylhexosamine, N-acetylglucosamine, or sialic acid on proteins and glycolipids of host cells.

Table 1
Table 1
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When sufficient numbers are present, some bacteria produce a biofilm which can establish and also protect their toehold and provide resistance to disinfectants, antibiotics, and elimination by host protective responses.16 The biofilm allows the bacteria to survive in hostile environments and colonize areas that would otherwise not support their growth. The biofilm is an adhesive matrix of polysaccharide that binds the cells to each other as well as to the surface. Formation of the biofilm is signaled by a critical concentration of a small molecule which is reached when there is a quorum, certain number of bacteria, each releasing the signal molecule. For example, Pseudomonas aeruginosa establishes a biofilm in the lungs of cystic fibrosis patients. Each of these bacteria signals the group by the release of an acylated homoserine lactone. The molecule is transported into the cell, and sensors activate the production of the biofilm components when this molecule reaches a critical concentration.17

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Weapon Launching Systems

Both gram-positive and gram-negative bacteria have systems for secreting proteins out of the cell.18 Gram-negative bacteria use several different mechanisms to transport proteins across the double hull of their cell wall, through both the plasma and outer membranes. Many of these proteins are weapons such as toxins, adherence, and penetration devices. These secretion devices either transport their cargo directly from the cytoplasm of the cell through both membranes to the outside of the cell in a single process or dump the cargo into the periplasmic space (described above) where it awaits transport across the outer membrane (Fig. 2).19

Figure 2
Figure 2
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The type I secretion system delivers proteins from the cytoplasm through the plasma membrane, peptidoglycan, and the outer membrane. The system consists of 3 proteins, a specific outer membrane protein, an ATP-binding protein (this is an ATP-binding cassette transporter system), and a membrane fusion or adaptor protein. This type of system is used to secrete toxins, degradative enzymes, and other proteins. Some examples include E. coli alpha-hemolysin, Pasteurella haemolytica leukotoxin, and P. aeruginosa protease.20

The type II system takes protein cargo that is released into the periplasmic space and transports it across the outer membrane. The type II system is composed of at least 12 proteins that form a cytoplasmic component, an inner membrane component, and an outer membrane pore.21 The type II system is used by V. cholerae to secrete its toxin and by other bacteria to secrete toxins and hydrolytic enzymes.22

The type IV secretion system is similar to the type III in structure and is used primarily for the cell to cell transfer of DNA, for example during conjugation. Several pathogens can also use the type IV injection capability to shoot proteins out of the cellular envelope and into eukaryotic cells. Bordetella pertussis uses the type IV system to secrete part of its toxin, PTx.23 Helicobacter pylori uses a type IV secretion system to inject Cag A into gastric epithelial cells.24

The type V secretion system, also known as an autotransporter system, is the simplest of these secretion systems. In this system, the proteins secrete themselves. The proteins to be released have 3 domains, an N-terminal signal, a passenger, and a C terminal or beta domain. The signal peptide is the ticket for the "sec" pathway to secrete the protein into the periplasmic space. The protein then associates with the outer membrane and forms its own pore by making a beta barrel structure with its C terminus. After these proteins are secreted through the pore, the C-terminal beta barrel of the protein is then cleaved off and retained in the outer membrane or cut loose into the extracellular space.25 The immunoglobulin A1 protease of N. gonorrhoeae is secreted by this autotransporter secretion pathway as are proteins from yersinia species, B. pertussis, and other bacteria.

The type III secretion device is gram-negative bacteria's ultimate weapon secretion system. Several gram-negative bacteria, including yersinia species, P. aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic E. coli (EPEC), and also chlamydia species, use this nanobiomachine to inject molecules into their eukaryotic host cells to facilitate infection.26,27 The type III secretory device, actually looks and works like a syringe (see Fig. 2, Inset). Related in structure and function to flagella, this device has a protein ring to anchor itself in the cytoplasmic membrane, a tube that traverses the peptidoglycan and the outer membrane and then an injector tip which has proteins that spear through and form a pore in the eukaryotic membrane. Unfolded or folded bacterial proteins can then be injected into the eukaryotic cell.28 There are 2 basic types of proteins that are secreted by the type III system, pore forming factors, and effector molecules. The pore-forming factor or translocator is secreted first and forms a pore on the host cell membrane. The effectors are then able to be injected into the host cell cytosol via this pore. The effector proteins have a wide array of activities and functions, some are able to activate actin rearrangements, such as membrane ruffling, to facilitate uptake and invasion, other proteins promote the intracellular survival and replication of the bacteria or promote apoptotic death of the host cell. The proteins may also be the components of a pedestal which is used for adhesion.29

Yersinia uses the type III secretion system to inject proteins30 which inhibit normal phagocytosis by macrophages by injecting a protein with tyrosine phosphatase activity (YopH) which dephosphorylates proteins necessary for normal phagocytosis, cause a collapse of the cytoskeleton by disrupting actin microfilaments which kill the cell (YopE), and cause other mayhem. Most incredibly, EPEC secretes a "portodocking system" through the type III system. After puncturing the host cell, several proteins are injected which embed themselves and stick out of the host membrane to act as mooring posts to facilitate the adhesion of the bacteria to the gut cell surface. This process can be viewed as an animation at the Howard Hughes Institute website (available at: http://www.hhmi.org/biointeractive/). Shigella uses the type III device to tickle epithelial cells into endocytosing the bacteria by injecting proteins which will rearrange actin and induce ruffles on the cell surface. The bacteria then will lyse the endocytic vacuole to escape into the cytoplasm.31 Salmonella uses a similar mechanism as Shigella to facilitate its own uptake into what is called an SCV or salmonella-containing vesicle in which it can survive and replicate within the cell.32

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Invasion

Invasion of host cells provides bacteria with a vehicle to spread to other parts of the body, find a niche, gain access to an "open kitchen and bar" for metabolites, and/or to obtain a hiding place from host innate and immune protections. The chlamydia and rickettsia live inside cells as obligate intracellular parasites because they cannot make sufficient energy for survival and must tap into the host cell for their ATPs (energy). Brucella, pasteurella, francisella, neisseria, and the mycobacterium use the inside of their host cell as a hiding place from antibodies and neutrophils. Salmonella, shigella, and E. coli invade the cells lining the GI tract as the first step in their spread to other tissues in the body.

As indicated above, after adhering to the target cell, the bacteria either trick or promote the cell into internalizing them. Tight binding to the appropriate receptor will trigger protein kinases and other signals that rearrange the cytoskeleton to promote phagocytosis. A bacterial protein called invasin is found on the surface of yersinia species (enterocolitica and pseudotuberculosis) and stimulates engulfment. Invasins on the surface of the bacterial cell bind to host cell surface receptors which indirectly trigger actin production and the rearrangement of the cytoskeleton to promote phagocytosis.

Once a bacteria is phagocytized, especially if it is in a macrophage, it must have a mechanism for avoiding or inactivating the killing mechanisms of the phagolysosome to establish its base of operations within the cell. Mycobacteria and legionella prevent the acquisition and inhibit the degradative enzymes of the lysosome by blocking the fusion of the phagosome with the lysosome and by preventing the acidification of the vesicles, inhibiting the action of degradative enzymes. The waxy layer surrounding the mycobacteria also protects mycobacteria from the actions of the lysosome. Listeria and shigella are able to escape from the lysosome.

Once inside the cell, shigella and Listeria monocytogenes can propel themselves into adjacent cells. Both bacteria have a protein which causes cellular actin to polymerize. The shigella protein is at the front of the bacteria causing the actin to form a spear which penetrates the adjacent cell providing a path for shigella to enter the cell.33 The listeria protein (Act A) is at the rear of the bacteria and the polymerization of the actin propels the bacteria into an adjacent cell as if on the top of a battering ram.33

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Command and Control

Weapon systems require coordinated control for proper function at the appropriate times. Many of these systems consist of multiple components which assemble into complex structures, such as the type III secretory device, or require coordinated synthesis of toxins and related molecules. In many cases, these proteins are encoded by genes that are grouped together in chromosomal regions termed pathogenicity islands that can be controlled by a single promoter responsive to a single activator. The activator may be a molecular or physical (eg, temperature) trigger which activates the expression of the entire group of genes. One such activator is DNA adenine methylase (Dam), a "master controller of many virulence factors."34 Another recently described controller for enterohemorrhagic E. coli is adrenaline (described earlier). The bacteria can sense when they are in the GI tract and also when the individual is stressed enough to be susceptible to infection. Genes for adhesins, invasins, and exotoxins may also be clustered together in pathogenicity islands. These pathogenicity islands can even be transmitted to other bacteria as a unit by genetic conjugation through pili or on plasmids. Some salmonella species possess 2 pathogenicity islands termed salmonella pathogenicity islands 1 and 2, both of which contribute to virulence via type III secretion. Salmonella pathogenicity island 1 encodes a type III secretion system that facilitates injection of effector proteins which target the actin cytoskeleton and expedite bacterial invasion. Salmonella pathogenicity island 2 works after invasion and encodes a second type III system which translocates proteins that enable the bacteria to survive and replicate within its vacuole inside a macrophage.35

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Achilles Heel
Disruptors and Inhibitors

Despite or because of the importance of the cell wall, it is a prominent Achilles heel of the bacteria. As for a submarine or a spaceship, a small break, a leak, a weakening of the outer shell will subject the bacteria to death and destruction. For this reason, other microbes target the cell wall constituents with antibiotics as do host innate protections. For example, the outer membrane is held together by hydrophobic and divalent cation interactions. Bacillus polymyxa releases the cationic lipopeptide antibiotic, polymyxin, which inserts into the membrane and displaces the divalent cations of the outer membrane disrupting its structure.36 Colistin, a close cousin of polymyxin, and daptomycin act in the same way. These antibacterials also disrupt the plasma membrane of gram-positive bacteria. Animals, from the honeybee to human, produce antibacterial peptides that disrupt gram-positive and gram-negative membranes. Mellitin is produced by bees and alpha and beta defensins are produced by human and animal leukocytes and epithelial cells to protect the mouth, lungs, urinary and GI tracts.37-39

The cell wall is also the target for many disinfectants which have detergent action. Detergents can solubilize the membrane killing the cell. Bacteria that live in the GI tract must have protections from the detergent action of bile acids. The outer membrane of gram-negative bacteria and the tightness of the peptidoglycan of some gram-positive bacteria provide this protection. Streptococcus aureus and other gram-positive bacteria develop resistance to common house cleaners (eg, pine oil) by building a thicker and tighter peptidoglycan. This also results in resistance to vancomycin.40,41

The peptidoglycan is another very vulnerable target. The cell wall is constantly being remodeled with a balance between synthesis and cleavage of the structure. Lysozyme cleaves the glycan portion of peptidoglycan and facilitates the turnover of peptidoglycan for the bacteria, but lysozyme is also secreted into mucous and tears to provide protection from gram-positive bacterial infection of humans. Inhibitors of peptidoglycan synthesis disrupt the balance between synthesis and turnover and when synthesis is inhibited, the peptidoglycan becomes degraded. In addition, bacterial growth and cell division are coupled to the expansion of the cell wall. β-Lactam antibiotics, vancomycin, and bacitracin inhibit peptidoglycan synthesis.

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Innate and Immune Homing Signals and Host Antibacterial Weapon Systems

The host has developed innate and immune radar systems to detect and home in on the structures of the cell wall components. This is especially facilitated by the repetitive nature of many of the structures and molecules on the bacterial surface.

The alternative and the lectin pathways of the complement system are activated by the repetitively charged nature of bacterial surfaces. The system drills holes in the membrane or attracts and promotes the uptake and killing of the bacteria by neutrophils and macrophages. Gram-negative bacteria are protected from complement action by a long O antigen on LPS, and the M protein of S. pyogenes binds and activates an inhibitor of complement. Similarly, a capsule provides protection from complement.

Peptidoglycan, teichoic acid, LPS, and flagella consist of distinctive repetitive patterns of molecules which provide "pathogen-associated molecular patterns" that can be recognized by host toll-like receptors (TLR).11 Human TLRs are a family of 10 different receptors that bind to pathogen-associated molecular patterns and then initiate a cascade of molecular events leading to the activation of nuclear factor κB-controlled genes for antibacterial inflammatory responses including cytokines (interleukin [IL] 1, tumor necrosis factor α, and IL-12), adhesion proteins, inducible nitrogen oxide synthase, and other protections. A cascade of protections becomes initiated including the production of interferon gamma by natural killer, natural killer T, and T cells, which will activate macrophages. Activated, also known as angry, macrophages are more efficient killers of bacteria. Peptidoglycan, bacterial lipoproteins, teichoic acid from gram-positive bacteria, lipoarabinomannan, and phosphatidylinositol dimannoside from mycobacterial cell walls activate TLR-2. The lipid A portion of LPS is the active component of endotoxin and is the most potent initiator of the TLR system. It activates TLR-4 and also binds to an LPS binding protein to trigger CD14 protective responses on macrophages. Lipopolysaccharides shed by bacteria or in bacterial debris are potent activators of TLR responses and lead to sepsis. Neisseria meningitidis readily shed their lipooligosaccharide which promotes innate responses resulting in increased vascular permeability and fever characteristic of meningococcal disease.11

The outside structures of the bacteria are also prime targets for antibodies. These smart bombs home in on the cell wall structures, activate complement and also target the bacteria for engulfment and killing by macrophages and neutrophils.

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CONCLUSIONS

As described in the introduction, bacterial cell walls have many things in common with the shells of submarines and space stations. Unlike these metallic structures, bacterial cell walls are a living and adapting structure, and their development has been funded and directed by Mother Nature. The sophistication of the packaging, attachment mechanisms, energy sources, and weapon systems have had the opportunity to evolve over the eons of bacterial existence. Improvements have developed by mutation and the selection processes forced upon each of the bacteria by their niche environment. These changes have been sped up by the promiscuous sharing of genetic cassettes, including pathogenicity islands. Unfortunately, the ability to genetically adapt to their niche and to the hazards of their environment makes it increasingly difficult for humans to control their outgrowth and in many cases, the disease consequences that result.

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ACKNOWLEDGMENTS

The authors would like to thank John Bower, MD, and Jonathan Brammer, BS, for their discussions regarding this manuscript.

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REFERENCES

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