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P.aeruginosa is a bacterium that needs to adhere and colonise on a host to establish an infection. Type IV Pili (T4P) mediates this adherence, and is also associated with the bacteria's twitching motility. PilY1 is gene within T4P which is involved with function of pili extension and retraction. Orans et al. analysed the crystal structure of PilY1 C-terminal domain, and revealed two major functional domains; a modified beta-propeller and a unique calcium binding loop. It was found that the CTD of PilY1 existed in two forms; calcium bound, which inhibits pilus retraction and, calcium free in which pilus retraction can freely proceed. Site directed mutagenesis was used to distinguish the exact amino acids involved with the twitching motility. They then hypothesised that the conversion between these two different states shows that PilY1 acts as a calcium dependant regulator for pilus retraction and extension, and therefore is responsible for the T4P mediated twitching motility seen in P.aeruginosa.
The location of PilY1 within Pseudomonas aeruginosa
P.aeruginosa is a gram negative bacteria which inhabits a range of environments. The bacteria are significantly opportunistic in immune-compromised or immune-suppressed individuals and can be life threatening due to being highly resistant to antimicrobials (Rehm, 2008) . In order to establish an infection the bacteria need to adhere and colonise on the host, this is adherence is partly mediated by type IV pili (T4P). T4P are also involved with twitching motility which is a mode of surface translocation; a kind of motility for bacteria across solid surfaces (Bohn, et al., 2009). T4P is one of two components of the P.aeruginosa bacterium responsible for motility, the other being a single polar flagellum which gives motility in liquid environments. (Murray & Kazmierczak, 2008).
T4Ps "are thin filaments that extend from the poles of a diverse group of bacteria, enabling them to move at speeds of a few tenths of a micrometer per second" (Skerker & Berg, pg1, 2001). The filaments consist of a single protein subunit called PilA (Bohn, et al., 2009) which is not only a structural protein but is also involved with a adhesion. This subunit is incapable of self assembly and repeated polymerisation and depolymerisation involving additional proteins such as PilY1 is required to achieve pilus extension and retraction (Orans, et al., 2010). According to Jarrell and McBride in 2008 "at least two ATPase enzymes with competing activities are involved in these processes. PilB is required for pilus extension, whereas the related protein PilT is involved in pilus retraction" (Jarrell & McBride, pg 471, 2008). When studied by Orans et al in 2010 it was found that if PilT was mutated then pilus fibres formed could no longer retract and hence the twithcing motility was lost (Orans, et al., 2010). The pilY1 protein is found to be localised to the outer membrane of the bacteria though which the pilus extends and retracts (Bohn, et al., 2009).
The structural and functional domains of PilY1
PilY1 is a protein that is involved in the extension and retraction of the pilus in P.aeruginosa bacterium. The protein has two major functional domains which are shown in figure 1. The recent studies conducted by Organs, et al. in 2010 predicted that the PilY1 protein would exhibit a Î²-propeller fold in the C-Terminal Domain (CTD). Upon crystallisation of PilY1, the structured 505 residues in the CTD of the protein revealed a modified beta-propeller. This modified beta-propeller differed to the predicted model as it only consisted of seven blades rather than the expected eight. The modified domain was found to consist of thirty one beta
Figure 1. Strucuture of the C-terminal domain of P. aeruginosa PilY1. The PilY1 structure reveals a modified beta-propeller fold and novel EF-hand-like calcium-binding domain. Figure adapted from Orans et al. 2010.
strands and nine alpha helices. Out of the seven blades, blade I to IV very much represented the predicted model; however there is a deviation in blades V through to VII. There is a protein strand (beta-20) which was not expected and contains a short motif of an alpha helix and a beta turn beta, beta-20 is shared between blade V and VI. The seventh blade, blade VII is shorter than first thought and contains only three short strands (Orans, et al., 2010).
Organs, et al. also discovered a unique Calcium binding loop within PilY1 which is very comparable to a canonical EF-hand binding site, as can be seen in figure 2. The PilY1 loop, although very similar to the EF hand in the fact that it has seven points that bind to a calcium ion, seems to be more efficient in the fact that it only employs a stretch of nine amino acids to achieve calcium chelation. Where as a typical EF hand, utilises twelve residues between two alpha helices.
Figure 2. Superposition of the nine-residue calcium-binding site of PilY1, (green), on the canonical 12-residue site in human calmodulin, (cyan).
The regulatory role of PilY1
PilY1 is essentially a cluster of genes that includes pilY2, pilX, pilW, pilV, and pile, that codes for pilus biogenesis ( (O'Toole & Kolter, 1998). It has been found that PilY1 is responsible for other control processes, including the expression of lipase LipC (Bohn, et al., 2009). In the structural data observed by Orans, et al., 2010, it came to light that PilY1 works in a calcium dependant manner and the binding of calcium prevents pilus retraction. It was noted however that calcium binding did not effect the overall sturcture of the protein, but has an "important role in several aspects of the pilus biogenesis and regulation" (Orans, et al., pg 1069, 2010).
Interaction with Calcium ions
The Calcium ions involved with the regulatory function of the PilY1 protein seem to bind at the unique calcium binding loop which is similar to an EF hand. This binding is essential for normal pilus function and it regulates the function of the pilus, and therefore the twitching motility of the bacteria, by antagonising the activity of PilT (protein Involved with pilus retraction). The ion is bound to the protein with only seven contacts, including one from a water molecule and tone from an acidic side chain.
Similarities to other proteins
When looking at the structure discovered by Orans et al., 2010 there seems to be a significant sequence conservation between PilY1 and other pathogens with retractile T4Ps. It seems T4P biogenesis requires dozens of proteins to be conserved across a bacterial species. The PilC and PilY homologues from Neisseria meningitides and P.aeruginosa play a common role in the biogenesis of T4P and so the structure of was analysed to determine if the C terminal residues 550 in PilY1, like PilC were involved in adhesion, and this turned out to be correct.
When taking a close look at the modified beta propeller within the CTD of PilY1, and using the Protein Data Bank to compare, according to Orans et al., the modified beta propeller blades I-IV align very well with the blades of the canonical beta propeller enzyme, from Comamonas testosteroni, called quinohemoprotein alcohol dehydrogenase.
Also the first four blades of the modified beta propeller are very comparable to the beta propeller of the WD40 protein WDR5. The first four blades align well, but it seems that the point on PilY1 which corresponds with where WDR5 interacts with a peptide, is blocked showing that PilY1 doesn't utilise the canonical WD40 binding site for protein-protein mediated reactions. The binding site is blocked by PilY1's helices four and seven, these helices are thought to be conserved from the Neisseria PilC proteins.
Examination of the unique calcium binding site on the CTDs of pilus biogenesis proteins indicates conservation in PilY1s, PilCs and PilY1 orthologues in other bacteria that make use of T4P. The exception however seems to be the replacement of Asn-853 in Neisseria PilC proteins with an aspartic acid, shown in figure 3.
Figure 3. Sequence conservation in the calcium-binding sites of the PilY1 homologues PilC1 and PilC2 from Neisseria meningitidis (N.men.) and N. gonorrhoeae (N.gon.) (Orans, et al., 2010).
In order for Orans et al. to understand whether or not calcium binding to the CTD of PilY1 effected the structure of the protein, firstly the bacteria was mutated. The mutation was the change of the calcium chelating Asp-859 to an alanine in order to eliminate calcium binding. The calcium was the removed by Chelex 100.
Chelex 100 is a competitive calcium ion binding assay, that is rapid and thorough, and is composed of styrene divinylbenzene copolymers with paired iminodiacetic (IDA) ions as the functional group (Figura & McDuffie, 1977). The IDA bind with the calcium acting as chelating groups successfully removing the calcium ions from the protein without having any effect on the protein concentration or function (Bio-Rad, 2006).
An advantage to this ion extraction technique is that automated procedures eliminate the risk of contamination, and even if non automated procedures are used there are fewer tube changes than traditional extraction methods (Bio-Rad, 2006). A disadvantage to Chelex-100 is that the conditions used in the extraction can be harsh and temperatures can get quite high leaving a small chance of protein denaturation (Hoff-Olsen, et al. 1999). Also there is no purification step with this procedure and some samples may need this is there to eliminate the risk of contamination, so a purification step may need to precede the use of Chelex-100 (Comey, 1994)
To find out if the structure of the protein had been affected by the removal of the specific calcium binding, the structure was measured sing circular dichroism.
Circular Dichroism (CD) is a phenomenon in which left circular polarised light and right circular polarised light are absorbed differently by a certain material. The difference results in elliptical polarised light being produced (Szilágyi, 2002). CD is a powerful structural biological method for distinguishing the secondary structures of proteins (Whitmore & Wallace, 2007).
In order to produce a spectrum from CD, a molecule must contain a chromophore and be chiral, since amino acids have both these characteristics they can be analysed by CD. The CD spectrum differ due to the chromophore being in a different environment depending on the amino acid, hence secondary structure can be determined. The side chains on amino acids cannot obtain a spectrum since they are not in a chiral environment, but of course if a protein is folded then the chromophore will be in a chiral like environment and so folded state of protein can be determined.
The spectra produced from a protein can indicate structural information from a single molecule. This information is then compared to a spectrum taken from a protein for which the secondary structure is already known (Whitmore & Wallace, 2007).
In the study conducted by Orans et al. CD was completed in the presence and absence of calcium and when looking at the results (figure 4) it is clear that the structure was not affected. Therefore it can be concluded that calcium binding is not involved with the overall structure of PilY1 CTD (Orans, et al., 2010).
Figure 4. Circular dichroism spectropolarimetry wavelength scans for purified P. aeruginosa PilY1 C-terminal domain proteins in the presence and absence of
Calcium (Orans, et al., 2010).
Site directed mutagenesis
In order to reveal the function of binding and release of calcium by PilY in P.aeruginosa, the biological analytical technique of site directed mutagenesis was utilised. This technique was developed in the seventies and its potential was apparent from onset:
"This new method of mutagenesis has considerable potential . . . to define
the role of. . . origins of DNA replication, promotes and the sequences for
ribosome-building sites. . . . specific mutation within protein structural
genes . . . will allow precise studies of protein structure-function relationships."
Hutchison et al. (1978)
Site directed mutagenesis requires a DNA primer with the amino acid change to be synthesised. This chemically synthesised oligoneucleotide is then hybridised with a single stranded template (M13 or a plasmid), then extended using DNA polymerase and deoxyneucleoside triphosphates then using T4 DNA ligase, the mutagenic primer ligates to the ends of the new strand. This double stranded DNA is then inserted into a competent E.coli and once cloning is undergone, both mutant and wild type protein will be produced (see figure 5). As long as the frequency of mutants produced is above fifty percent then they can be identified by directly sequencing a few clones. If there are few mutants produced then hybridisation screening is the easiest way to identify them. Hybridisation screening can be done either by using a probe that is a perfect match to selectively hybridise the mutant strain, or hybridise for all strains then selectively wash away the wild type (non mutated) strain. Clones remaining on the probe may then be sequenced to confirm the mutation. It is also necessary to sequence the whole protein produced to ensure there are no mutations elsewhere in the sequence (Carter, 1986).
Figure 5. Site directed mutagenesis (Nobel-Media, 2010).
The generation and characterisations of the mutations within a protein is essential when trying to correlate mutations with the phenotype of a protein. Site directed mutagenesis is one of the most widely used techniques for this and has been extremely successful through our molecular biology and biotechnology (Primrose & Twyman, 2006). This technique (site directed) replaced the original method of mutagenesis as the mutation position and sequence can be completely specified. Also combinations of mutations can be easily generated (Lichter & Hagar, 1987). There are however a few disadvantages to this technique that need to be considered. Firstly the early methods used single stranded plasmids and gave low yields of mutated species. Techniques were developed that select against wild types to give higher numbers of mutants produced but these developments were extremely costly and so impractical. Since then the development of the Polymerase Chain Reaction (PCR) has meant the procedure although quite timely is not as expensive. Use of PCR means, that mutations can be introduced at any point of the protein, whether restriction sites are present or not.
Site directed mutagenesis was used by Orans et al. to determine which amino acids were specifically involved with the twitching motility function. They found that when the PilY1 gene was removed completely from P.aeruginosa that twitching motility was lost, and the strain was devoid of surface T4P. When PilY1 was reinserted the twitching motility and pilus production was restored, proving that the PilY1 gene is involved with the production of T4P and the twitching function. It was also seen that when a mutated PilY1 gene carrying the substitute D859A, was inserted into P.aeruginosa lacking PilY1, there was dramatically reduced T4P production and twitching was obviously lowered compared to wild type P.aeruginosa. Which suggests that the aspartic acid removed may play a part in pilus function (Orans, et al., 2010).
To discover whether or not the defect in the D859A mutation was affected by pilus extension or retraction, the D859A mutant was expressed in a strain of P.aeruginosa lacking PilT (the gene responsible for pilus retraction). These bacteria had normal levels of T4P production and twitching motility, showing that the defects were caused by a PilT mediated reaction. When PilT was present the D859A mutant protein was unable to antagonise pilus retraction and so Orans et al. hypothesised that "the calcium-binding site in the PilY1 CTD is necessary for normal pilus function by antagonizing the retractile activity of PilT" (Orans, et al., 2010, p. 1067)
The work carried out by Orans et al. 2010 clearly indicates that calcium binding is an important factor which regulates the twitching motility of P.aeruginosa. Switching between the calcium bound and calcium free state in the wild type P.aeruginosa is necessary for pilus extension and retraction. Other proteins have conserved sequences to PilY1 such as PilC in neisseria and other homologues. Orans et al. 2010 predicted that in these proteins calcium binding may also be a key aspect of pilus biogenesis and regulation.
The structural and functional information revealed by Orans et al. 2010 gives a insight into the complexity of T4P regulation and it seems that there hypothesis for the protein P.aeruginosa may also be true for a range of pathogenic bacteria which employ the same twitching motility.