DISCUSS THE STRATEGIES THAT ARE USED BY BACTERIAL PATHOGENS TO EVADE HOST ANTIBACTERIAL PEPTIDES AND PHAGOCYTES

“DISCUSS THE STRATEGIES THAT ARE USED BY BACTERIAL PATHOGENS TO EVADE HOST ANTIBACTERIAL PEPTIDES AND PHAGOCYTES”
The mammalian immune system has two major components; the innate and the adaptive immune system. The innate system is usually the first line of defence and it acts as a strong roadblock to invading bacterial pathogens through direct mechanisms such as inflammation, phagocytosis, complement system activation, antibacterial peptides and indirectly by activating adaptive response through antigen presentation (Radtke and O’Riordan, 2006). However, in order to successfully carry out and maintain infections, bacterial pathogens have evolvedtodevelop some strategies for the invasion, resistance or completely avoid these hosts defence mechanisms. By doing this, they will be able to damage cells and even multiply in certain areas they normally would not be able to(Cossart and Sansonetti, 2004).These strategies, particularly those employed in the evasion of antibacterial peptides which are small cationic effector molecules such as Defensins, Cathelicidins and Thrombocidinsand phagocytes; white blood cells capable of engulfing bacteria, dead cells and foreign particles(Boman, 2003) will be discussed in details.
EVASION OF ANTIBACTERIAL PEPTIDES
In the attempt to occupy certain areas of their host’s anatomy, bacterial pathogens will be exposed to antibacterial peptide-mediated host defences at some point. Although, these host defences may succeed in clearing the infection, some of the pathogens are able to evade thisline of defence in various ways.
Alteration of cell surface.Antibacterial peptides such as defensin and cathelicidins carry out defence actions based on their ampiphatic structure and charge. As most microbes present a negative charge on their cell surface, antibacterial peptides can easily be attracted to them because of their cationic property (Gutsmannet al, 2001).On successful accumulation of high concentration of these peptides on the surface of the microbes, ion channel or aqueous pores are formed via the intercalation and accumulation of their hydrophobic faces in the cytoplasmic membrane which is the main site of action. This consequently leads to the death of the microbe by hypo-osmotic lysis (Oren et al, 1999). To reach the cytoplasmic membrane however, antibacterial peptides must pass through the thick cell walls of gram-positive bacteria which contain cross-linked polymers of teichoic acid or lipoteichoic acid and peptidoglycan or the outer membrane of gram-negative bacteria composing of lipid A, core polysaccharides and O-side chain known as lipopolysaccharide (LPS) (Nizet, 2006). In Staphylococcus aureus, a gram positive pathogen, resistance to antibacterial peptides occurs through the specific alteration of the teichoic acid as shown in fig. 1. The presence of phosphate groups in great number in teichoic acid contributes to its polyanionic properties and this negative charge is neutralized in the cell wall of S. aureus via the esterification with significant amount of D-alanine. This consequently causes the repelling of antibacterial peptides before getting to the site of action (Peschel, 2002).Peschelet al, (1999) confirmed that D-alanylation of teichoic acid in S. aureus is achieved via gene products of dlt operon. They showed that mutation in the dltoperon led to increase in cell surface negative charge and became highly susceptible to killing by defensins and cathelicidins while the wild type overexpressed the dlt gene and was more resistant. Mutations in dltA gene has also been shown by Poyartet al, (2003) to cease D-alanylation of teichoic acid in Group B Streptococcus which made them prone to killing by human defensins and same effect was seen in Bacillus subtilis in the study by Cao and Hellman, (2004). In gram negatives, alteration in the lipid A of lipopolysaccharides is the main mechanism of antibacterial peptide evasion with regards to cell surface modification as it reduces the negative charge. In Salmonella entericaSerovarTyphimurium, lipid A alteration resistance is caused when 4-aminoarabinose (Ara4N) is added to phosphate group of the lipid A backbone, fig. 1 (Nizet, 2006). Similarly, in Proteus mirabilis, addition of Ara4N to lipid A causes antibacterial peptide resistance and a study by McCoy et al, (2001) showed that mutation causing loss of Ara4N made P. mirabilis more susceptible to antibacterial peptide killing. In addition, antibacterial peptide in Salmonella has also been associated with two component regulatory system (PhoP/PhoQ) that controls pagP genewhich consequently helps in the acylation of lipid A. Mutants have been shown to have higher outer membrane permeability to antibacterial peptides compared to the wild type (Guoet al, 1998). Overall, a very usual mechanism of antibacterial peptide evasion either by gram negative or gram positive bacteria is by alteration of the normal negatively charged cell surface components with positively charged one so as to avoid the cationic antibacterial peptides reaching the cytoplasmic membrane.

Fig. 1: Resistance to antibacterial peptides through cell surface modification. Normal negatively charged cell surface becomes more positively charged causing evasion of the effects of cationic peptides.
External trapping of antibacterial peptides. Some pathogenic bacteria can evade antibacterial peptides killing by binding or neutralizing them either by direct actions of bacteria secreted protein or by indirectly by inducing the binding of these peptides to the hosts cell surface. This reduces the actual quantity of antibacterial peptides reaching the bacteria to carry out the killing (Nizet, 2006). Staphylococcus aureus releases staphylokinase (SK), a protein capable of binding to α-defensins produced by neutrophils. Jin et al, (2004) showed that binding of SK to α-defensinsinhibits their action. They suggested that binding of antibacterial peptide and the action of dlt on cell surface modification helps staphylococcus aureus successfully carry out infections. Bacterial inactivation of antibacterial peptides can occur by taking advantage of negatively charge proteoglycan molecules on the epithelial cell surface of the host. Group AStreptococcus and Pseudomonas aeruginosa releases certain proteases which breaks down decorin, a cell surface proteoglycan and dermatan sulphate is released. Inactivation of human α-defensin occurs when the free dermatan sulphate binds to it (Schmidtchenet al, 2001).
Efflux of antibacterial peptide.Another mechanism that has been developed by bacterial pathogen against antibacterial peptides is the efflux. Shafer et al, (1998) showed that evasion of antibacterial peptide is achieved by an energy-dependent efflux system namedmtrin Neisseriagonorrhoeae. Other studies show that mtrCDE complex removes antibiotics, dyes and detergents which suggest that the mechanism protects pathogens against other stress causing stimuli (Bengoechea and Skurnik, 2000). In Yersinia, an antiporter of potassium is formed by RosA and RosB protein which is involved in the efflux of antibacterial peptide. The gene regulating these proteins is induced on exposure to antibacterial peptides and this suggests that Yersinia can manage to survive in an environment rich in antibacterial peptides (Stumpe and Bakker, 1997). These findings show that bacterial pathogens have evolved to develop structure-specific and energy-dependent mechanisms to evade host antibacterial peptides.

EVASION OF PHAGOCYTOSIS
Phagocytosis is another line of defence invading bacteria encounter in order to successfully infect their host. This is a receptor-mediated recognition and internalization of extracellular particles such as pathogens, debris or dead cells (Celli and Finlay, 2002). Professional phagocytes such as macrophages and neutrophils are able to express phagocytic receptors for the recognition, binding and eventual destruction of pathogens. Opsonin-dependent mechanism of phagocytosis involves Fcγ receptors which binds to substances with IgG on their surface or complement receptors (CR1, CR2 and CR3) that binds to particles expressing surface complement. Also, phagocytosis can be induced by different cellular receptors such as mannose receptors, type A scavengers and integrins that recognize and bind directly to surface molecular motifs on pathogens; this is regarded to as opsonin-independent phagocytosis (Aderem and Underhill, 1999). Phagocytosis however, can be seen as an obstacle or assistance to bacterial pathogens because they have evolved strategies to either survive in phagocytic cells or avoid the whole process as a whole.

Avoiding recognition by phagocytic receptors.In order to evade phagocytosis via Fcγ receptors, some bacteria pathogens such as Neisseria spp. and Staphylococcus aureus have evolved surface antigenic variation strategy in order to avert acknowledgement by specific antibodies. Neisseria spp. does this by altering the antigenicity of pilin, a component of pilus which is a cell surface molecule (Seifert, 1996). Staphylococcus aureus avoids Fcγ-induced phagocytosis by antibody interference. Binding of Protein A from S. aureusto the Fc region of IgGcauses prevention of the normal interaction with Fcγ receptors (Forsgren, 1983).Regarding complement-induced phagocytosis evasion, it has been established that some pathogenic bacteria such as Escherichia coli and S. aureus forms a capsule from polysaccharides on their surfaces which prevents complement deposition and consequently hinder phagocytosis (Silver et al, 1988; Thakkeret al, 1998). Similarly, Group A Streptococci is able to avoid opsonisation and killing by phagocytes through the presentation of cell wall anchored M-proteins(Fischetti, 1989). M-proteins have a binding site for C4b-binding protein (C4BP), a complement regulator and this enhances breakdown of C3 convertase enzyme of classical pathway as well as degrade C4b (Berggardet al, 2001). In addition, M-proteins are known to have regions for binding factor H, FH-like protein (FHL-1) and fibrinogen. FH and FHL-1 are associated with the regulation of complement activation via the alternative pathway and binding to M-proteins have been shown by Kotarskyet al, (2001) to control the deposition of C3-derived opsonins on bacteria surfaces leading to evasion of phagocytosis (fig. 2).

Interruption of cell signalling pathways.Recognition and phagocytosis of non-opsonised bacteria is achieved by professional phagocytes as they possess opsonin-independent receptors such as type A scavenger and mannose receptors. To avoid internalisation, contact dependent strategies must be employed by phagocyte-bound bacteria which must act to prevent complete phagocytosis. Specialised secretion systems such as type III secretion systems as shown in fig. 2, which are committed to deliver proteins (effectors) to cells of hosts are used by different gram negative bacteria pathogen to undermine hosts cell functions(Celli and Finlay, 2002). Type III secretory systems are congregated together from about twenty proteins to form a structure with resemblance to ‘molecular syringe’ (Hueck, 1998) and are created to convey effectors at the location of phagocytosis initiation leading to interruption of phagocytic signalling pathways triggered upon engaging receptors (Goosneyet al, 1999). In pathogenic Yersinia species for example, the evasion of phagocytosis by macrophages after the engagement of β1 intergrin receptor is by translocation of different host cells effectors that induce antiphagocytosis (Wiedemann, 2001).

Fig. 2: Different mechanisms of phagocytosis evasion. (a) GAS interference with surface complement deposition by C4BP binding, fibrinogen, FH and FHL-1 through expression of surface M-protein. (b) Inhibition of phagocytosis by bacteria pathogen using type III secretion system.

Escape from phagosome.Once internalised in phagosome, the fate of a bacteria undergoing phagocytosis is to be conveyed to the lysosome for destruction. However, microbes such as Listeria, Rickettsia and Shigella can avoid this by escaping from the phagosome to the cytoplasm (Gouinet al, 1999). Internalised Listeria in a phagosome expresses Listeriolysin O, a pore forming protein and this forms pores in the phagosomal membrane causing its degradation by allowing access of two Listeria-encoded C-type phospholipase. Once the membrane is degraded, the bacterium enters the cytosol and multiplies (Goebel and Kuhn, 2000).

Inhibition of phagosome-lysosome fusion.Bacterial pathogens also avoid being conveyed to lysosomes by inhibiting the fusion of phagosome and lysome. This strategy ensures the organism survives in the phagosome as well as blocking the activation of immune system through the presentation of antigenic peptides by class II MHC molecules (Pieters, 2000). Salmonella possesses Spl-2, another type of type III secretion system different from the one used in gaining entry into non-phagocytic cells and it is up-regulated on entry into a phagocyte (Cirilloet al, 1998). SpiC, a protein encoded by Spl-2 then effectively impedes the fusion of phagosome to lysosomes in macrophages hence distorting the process of phagocytosis(Uchiyaet al, 1999). Furthermore, a study by Vazquez-Torres et al, (2000) shows that some of the genes encoded by Spl-2 prevents the recruitment of NADPH oxidase to phagolysosome which effectively blocks the generation of superoxides, a precursor to antibacterial molecules.Also, the survival of mycobacteria in mycobacteria phagosomes for long periods without being transferred to lysosomes is due to the active recruitment of a host protein TACO (tryptophan-aspartate containing coat protein) which is released before phagosome fusion with lysosomes. Effective retention of TACO inhibits its transport to lysosome promoting its survival (Ferrari et al, 1999).

Transport to non-lysosomal organelles.Some pathogens can choose their phagocytic receptor carefully to evade their delivery into lysosomes. Organisms such as Staphylococcus aureus, Streptococcus pyogenesand Mycobacterium spp. are internalised through complement receptors which does not always lead to activating macrophage and up-regulation of their killing activity (Blystone and Brown, 1999). A primary ligand for complement receptor (C3b) is mimicked by the production of a molecule on the surface of mycobacteria which leads to complement evasion via complement receptor and consequently evade phagocytosis (Schoreyet al, 1997).

CONCLUSION
The mammalian immune system helps in the defence against pathogenic microbes through various means including the production of antibacterial peptides and phagocytosis. However, these microbes have developed various mechanisms to evade these host defences. Different studies in this pathogen-host interaction do not only help in the understanding of these mechanisms, but also help in the development of novel approach to fight them.

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