Bacterial adhesion: From mechanism to control - 3. Cell surface structure mediating bacterial cell adhesion(진행중)

 

 

 

3. Cell surface structure mediating bacterial cell adhesion

 

As described above, cell surface structures directly affect microbial adhesion to solid surfaces. EPS and proteinous cell appendages have functions for bridging between the cell body and a substratum. While EPS is important for development of biofilm structure rather than for cell adhesion, proteinous cell appendages are often essential for the initial interaction between cells and a substratum, and function as adhesin.

 

3.1. Polysaccharides

3.1.1. Lipopolysaccharide

Lipopolysaccharide is composed of lipid A, core polysaccharides, and an outermost region of O antigen units [66,67] and is important for bacterial initial surface adhesion [68]. The common antigen (A-band) polymer is composed of 10–20 repeating α -D-rhamnose units [69], while the serotype-specific antigen (B-band) is composed of di- to pentasaccharide repeating units [70]. The B-band has much longer polysaccharides than the A-band as well as a higher concentration of amino sugars, but a low concentration of sulfate and rhamnose [71].

 

The B-band has been shown to form hydrogen bonds of approximately 2.5 kT J (where k is Boltzmann constant and T is absolute temperature) each with mineral surfaces. It is suggested that 1000 or fewer such bonds are sufficient to anchor a cell firmly to a surface [72].

 

Simoni et al. [73] calculated the interaction energy between cells of Pseudomonas sp. and surfaces in a sand column and concluded that there was an energy barrier of several hundred kT J per cell at about 20 nm and that LPS hindered close contact of cells with surfaces. Consequently, they hypothesized that LPS would bind the cells to the surface from a distance of about 20 nm, and assumed that LPS could form hydrogen bonds with the substratum. Makin and Beveridge [74] showed that Pseudomonas aeruginosa PAO1 lacking B-band LPS demonstrated reduced adhesion to hydrophilic surfaces. Escherichia coli lacking LPS, as in P. aeruginosa, also had decreased ability to adhere [75]. However, because these LPS mutants were also defective in flagellum-mediated motility and type 1 pilus production, it was difficult to determine if the loss of LPS showed a direct or indirect effect on the cell adhesion [68].

 

3.1.2. Exopolymeric substances

EPS are classified into bound EPS that includes tightly bound EPS such as capsular polymer and loosely bound EPS such as slime, and into soluble EPS that includes polymers released into bulk water [76]. Biofilm structure is largely associated with the production of EPS, which provides the structural support for biofilms [77,78]. EPS accounts for roughly 50–90% of the total organic carbon of biofilms and is considered to be the primary matrix material of biofilms [76,79]. EPS is composed of primary polysaccharides and other macromolecules such as proteins, DNA, lipids and humic substances [80]. In this review, we mainly focus on polysaccharides, which are often investigated in regard to bacterial adhesion [81]. The EPS matrix that keeps the microorganisms together in biofilms is responsible for adhesion to a given surface [82,83]. Interactions between EPS and surfaces are caused by noncovalent bonds, such as electrostatic attraction and hydrogen bonds [84]. The binding force of these interactions is weak compared with a covalent C–C bond. However, these weak interactions are multiplied by the large number of binding sites available in the macromolecules, and the total binding force exceeds that of covalent C–C bonds [78,85]. The contribution of EPS production to bacterial adhesion and biofilm formation has been examined by mutation of genes involved in EPS synthesis. Danese et al. [77] showed that an E. coli strain defective in the production of colanic acid, a major EPS component, does not develop normal biofilm architecture. Since initial adhesion to the surface was not affected, however, colanic acid is unlikely to be involved in the first step of cell adhesion. Yildiz and Schoolnik [86] demonstrated that a wild-type strain of Viblio cholera produces a small amount of EPS and forms a thin biofilm, whereas spontaneous variants that produce an increased amount of EPS develop thick biofilms [87]. It has been reported that changes in gene expression are induced by contact of cells with a surface [88]. Davies et al. [89,90] demonstrated that the transcription of algC, a key gene involved in the biosynthesis of alginate required for the synthesis of EPS, is up-regulated three- to five-fold in adhered cells of P. aeruginosa compared with their planktonic counterparts within minutes of adhesion to surfaces. Garrett et al. [91] linked the down-regulation of flagellum synthesis with the up-regulation of alginate synthesis. Cells adjusting to an immobile life on surfaces lost their flagella and increased production of EPS. The molecular mechanisms contributing to the biofilm formation of Staphylococcus epidermidis have attracted significant interest. In this bacterium, polysaccharide and protein factors mediate cell adhesion to native polymers [92–94]. It was also shown that S. epidermidis can adhere to surfaces coated by a conditioning film containing extracellular matrix proteins such as fibrinogen or fibronectin [95]. Following the adhesion to the surface, polysaccharide intercellular adhesin (PIA) mediates the intercellular adhesion essential for accumulation of multilayered S. epidermidis biofilms [96,97]. The major PIA is a homoglycan composed of at least 130 β -1,6-linked N-acetylated and non-Nacetylated glucosaminyl residues and ones with positive charge [98]. PIA is synthesized by the icaADBC locus encoding the PIA synthesis apparatus [99]. Inactivation of the icaADBC locus by Tn917 insertion led to a biofilm- and PIA-negative phenotype of S. epidermidis [100].

 

3.2. Bacterial nanofibers

Many kinds of bacteria have filamentous cell appendages, which are several to tens nanometers in diameter and hundreds to thousands of nanometers in length, such as flagella and pili. These bacterial nanofibers have been shown to function as adhesin. Some bacterial nanofibers mediate cell adhesion to abiotic surfaces and are involved in biofilm formation. Others specifically bind to biomolecules on host cells and/or extracellular matrices (ECMs), such as collagen and fibronectin, and are involved in pathogenic infection.

 

3.2.1. Pili (Fimbriae)

The most well-known proteinous adhesin of Gram-negative bacteria is a hair-like nanofiber called a pilus or fimbria [3]. (The latter (pl: fimbriae) is a synonym for the former (pl: pili).) Pili are complexes consisting of several kinds of protein subunits, and a single protein subunit called a major pilin stacks by hundreds, being arranged into a helix to form a long rod-shaped body. The tip of a pilus contains several different proteins capped by the adhesin. There are several kinds of pili, which are varied in function, molecular structure, localization on a cell surface and mechanisms of secretion and assembly. A bacterial cell frequently has more than one type of pilus. Type IV pili in many Gram-negative bacteria including P. aeruginosa have been studied most in terms of the relationship with biofilm formation. This type of pili has been reported to be involved in both nonspecific adhesion to abiotic surfaces for colonization and biofilm formation, and specific binding to target molecules in hosts [101]. Type IV pili extend from the poles of a bacterial cell and mediate the movement of bacteria over surfaces without the use of flagella. These movements are known as social gliding in Myxococcus xanthus and twitching in organisms such as P. aeruginosa and Neisseria gonorrhoeae [102]. In fact, twitching motility of cells was visualized by direct observation of extension, fixation to a surface at the distal end, and retraction of type IV pili in P. aeruginosa under a fluorescence microscope [103]. These surface motilities are thought to contribute to enlargement and development of microcolonies and biofilms. Prepilin, a precursor of major pilin of type IV pili, is secreted through the inner membrane (IM) by the Secdependent pathway, and thereafter it is processed into a mature pilin by peptidase localized at the IM. The mature pilin subunits are assembled by the IM assembly complex towards the OM through the periplasm [3]. The assembled pili are translocated across the OM by a system related to the type II protein secretion system, where a multimeric OM protein forms a gated pore [3,101,104]. Type 1 pili and P pili are also well-known adhesive fimbriae, which are present on Gram-negative bacteria such as E. coli and Salmonella species. These types of pili are also composed of major pilin assembled into a nanofiber but are peritrichate unlike polar type IV pili. It has never been reported that these peritrichate pili are responsible for twitching motility. They are important virulence factors of pathogens for adhesion to host cells. They are secreted and assembled by the chaperone-usher pathway, in which a periplasmic chaperone and an OM assembly platform, the “usher”, are required (Fig. 3A) [3,105–107]. After translocation into the periplasm through the IM by the Sec-dependent pathway, subunits constituting the pili interact with the chaperone, which acts by a mechanism called donor strand complementation to allow proper folding of the subunits while simultaneously preventing premature subunit–subunit interactions. The chaperone-subunit complexes in the periplasm are targeted to the OM usher. Fiber assembly is thought to occur at the periplasmic face of the usher, concomitant with secretion of the fiber through the usher to the cell surface [108]. In E. coli strains, the structural pilus subunits of Type 1 pili and P pili are encoded by fim and pap gene clusters, respectively, together with the chaperone and the usher in the respective operons. Major pilins of type 1 pili and P pili are FimA and PapA, respectively. An adhesin subunit, FimH in type 1 pilus or PapG in P pilus, is located at the distal end of each pilus fibril. These pili are assembled in a top-down fashion, with the adhesin subunit incorporated first. These adhesin subunits bind specifically to target molecules such as sugars or glycolipids of hosts; for example, FimH is the mannose-specific adhesin [109]. Compared with Gram-negative bacteria, information about pili of Gram-positive bacteria is limited to a few groups such as Corynebacteria and Streptococci. They have largely gone unrecognized until recently, likely because they are extremely thin (2–3 nm) and difficult to observe [110]; however, these are reviewed by Scott and Zähner [111] and Telford et al. [112]. Pili of Streptococcus sp. have also been shown to have key roles in the adhesion and invasion process and in pathogenesis. Unlike Gram-negative pili, whose subunits associate via noncovalent interactions, Gram-positive pili consist of multiple, covalently bonded copies of a single backbone pilin, to which a few accessory proteins can be added. For their assembly, a transpeptidase enzyme called sortase is required. Sortase recognizes specific sequence motifs in the pilin subunits, which are secreted thorough the cell membrane by the Sec-machinery, elongates the pilus oligomer by progressive addition of subunits joined by intermolecular isopeptide bonds, and then tethers the entire assembly to the cell wall peptidoglycan (PG) [112–114].

 

3.2.2. Autotransporter adhesin

Recently, autotransporter adhesins (ATADs), which are nonfimbrial adhesins, have attracted considerable attention as a new class of virulence factors [115]. Unlike long, polymeric, hair-like pili, they are short monomeric or oligomeric nanofibers seen in Gram-negative bacteria. The designation “autotransporter” comes from the translocation mechanism known as the type V secretion system, which is one of the most widely distributed secretion systems among Gram-negative bacteria [106]. In this system, proteins are secreted out of cells without the mediation of other proteins. To date, two variations have been reported in ATADs; one is monomeric “conventional” autotransporters, and the other is a novel subfamily known as trimeric autotransporter adhesins (TAAs), which recent studies have identified [116]. In both types, after their secretion into the periplasm across the IM by the Secdependent system and procession of an N-terminal signal peptide, the C-terminal translocator domain is inserted into the OM and forms a β -barrel structure to form a pore. Subsequently, an internal passenger domain is secreted from the periplasm to the cell surface through the pore. Virtually all conventional types undergo a cleavage event that separates the passenger domain from the translocator domain [115]. This cleavage is often autoproteolytic, and the passenger domain remains either non-covalently associated with the cell surface or can be released into the extracellular milieu. By contrast, in all TAAs that have been examined to date, the passenger domain remains covalently linked to the translocator domain, which therefore becomes the membrane anchor domain [116]. The translocator domains in both conventional types and TAAs consist of 12 transmembrane β -strands, which are arranged in an antiparallel fashion in the OM and form a β -barrel pore with a hydrophilic inside and a hydrophobic outside. In the case of TAAs, one polypeptide has a C-terminal containing 4 β - strands and three of them are assembled into a 12-stranded β -barrel. The mechanism of translocation of the passenger domain through the translocator is still controversial, but it is independent of energy from ATP and there is a hypothesis that the folding of the passenger domain at the cell surface may provide a source of energy to drive secretion through the translocator [117]. The conventional ATADs contain an intramolecular chaperone domain, which is absent in TAAs [116]. As for TAAs, the passenger domains are supposed to be translocated across the pore as separate polypeptides, probably unfolded or partially folded as a folded trimer would be unable to cross the pore formed by a fused trimeric β -barrel (Fig. 3B). TAAs have been found recently in many Gram-negative pathogens, for example, Yersinia spp. YadA [118], Moraxella catarrhalis UspA1 and A2 [119,120], Haemophilus influenzae Hia and Hsf [121,122], Xanthomonas oryzae XadA [123], Neisseria meningitidis NadA [124], Bartonella henselae BadA [125] and Actinobacillus actinomycetemcomitans EmaA [126]. The architecture of TAAs is not a stack of a single major subunit as seen in pili, but a fibrous one that extends in the direction of the amino acid (a.a.) sequence of the polypeptide chain. Although TAAs vary in size from about 300 a.a. to over 3000 a.a., their molecular organizations have a common feature with variations; they form homotrimeric structures with a head-stalk-membrane anchor architecture in the orientation of the N-terminal toward the C-terminal, forming a “lollipop”-shaped structure [120,127]. Therefore, the length of the nanofiber depends on that of the polypeptide. Whereas the head and stalk domains are diverse and found in different combinations, the membrane anchor is the only domain that is homologous for all TAAs and thus defines this family. The membrane anchor contains four transmembrane β -strands that construct the fused trimeric 12-stranded β -barrel and a short coiled-coil segment, which is considered to occlude the β -barrel pore after export and folding of the passenger domain. The stalk domains of TAAs are fibrous, highly repetitive structures that are rich in coiled coils and extremely variable in length [127]. They function as spacers to project the head domains located away from the bacterial cell surface. For example, the stalk of Yersinia YadA, the prototype of TAAs, contains a right-handed coiled coil consisting of ten 15-residue repeats [128], whereas the stalk of Moraxella UspAs is formed by a left-handed coiled coil [120]. According to the common oligomeric structure of coiled coils, this family (TAA) is also called “Oca (oligomeric coiled coil adhesin) family” [129]. So far the head domain has been solved by X-ray crystallography only in YadA, Hia and BadA. Although both of YadA and Hia have the secondary structures that are rich in β -strands, their tertiary and quaternary structures are quite different [127]. All TAAs reported to date have a common function: adhesion to a host. The head domains are known to bind to ECM proteins such as fibronectin, collagen, and laminin, and to mediate self-agglutination of cells [115,127,130,131]. The high sequence diversity and distinct mosaic-like structures of TAAs have lead to difficulties in the annotation of their sequences. Recently however, Szczesny and Lupas [132] have developed a workflow referred to as daTAA for the accurate domain annotation of TAAs. This useful web-based annotation platform has already been used in the annotation and analysis of domain structures of complicated TAAs [133,134] and has the potential to contribute to the better understanding of the molecular structures that control cell-surface interactions.

 

3.2.3. Other unique nanofibers

The Gram-negative bacterium Caulobacter crescentus is one of the first colonizers of submerged surfaces in aquatic environments [135]. C. crescentus cells attached to a surface are capable of resisting washing with strong jets of water, suggesting that the attachment of single cells is extremely strong [136]. This bacterium has a dimorphic life cycle consisting of a stage as a swimming swarmer cell followed by a stage as a nonmotile sessile cell that has a polar adhesive nanofiber approximately 100 nm in diameter and several micrometers in length [137,138]. This adhesive nanofiber is called a “stalk” and tipped by holdfast, a polysaccharide adhesin that mediates the strong adhesion of the stalked cells to surfaces. The stalk is an extension of the cell envelope containing IM, PG, and OM, whereas the holdfast is composed of extracellular polysaccharides and additional components such as proteins [139]. Recently, Tsang et al. [140] reported that adhesion strength between the hold- fast and the substrate is at least 68 N/mm2, which is the strongest ever measured for a biological adhesive. Although polymers of N-acetylglucosamine, which are an important component in the holdfast, were revealed to play a critical role in the adhesive force [139], the chemical and biophysical basis for the impressive force of the holdfast adhesin is still unclear. Recently, a novel and unique peritrichate nanofiber with striking morphology was reported on cells of the archaea designated SM1 euryarchaeon living in cold, sulfidic springs [141]. This nanofiber called “hamus” (plural “hami”) is 7–8 nm in diameter, 1–3 m in length and consists of a “hook region” at the tip and a “prickle region” at the center stalk. The hamus filament possesses a helical basic structure, and at periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At the distal end of the hook region, the hamus carries a tripartite grappling hook with barbs. The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. Although the hamus was reported to be mainly composed of a protein of 120 kDa, there have been no further reports of molecular information about the hami. The authors showed that the hami mediate strong adhesion of single cells to each other and to surfaces of different chemical nature. They supposed that the hami might play a crucial role in the formation of the microbial string-of-pearls communities formed by SM1 euryarchaeon and other bacteria [142].