The Lipoproteins Of Streptococcus Pyogenes And Its Virulences Biology Essay

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Streptococcus Pyogenes bacterium is a worldwide known human pathogen, belongs to the Group A Streptococcus, GAS. It is Gram-Positive, non-motile, and spherical in shape which grows in long chains or pairs. S.pyogenes is distinguished from the other Streptococci by the presence of the Lance Field Group A carbohydrate found on its cell wall and it produces large zone of beta-hemolysis ( haemoglobin released when the erythrocytes are completely disrupted) when grown on a plate enriched in blood agar. Therefore it is known as the Group A (beta-haemolytic) Streptococcus (GAS).

Group A Streptococcal Infections is mainly caused by the S.pyogenes resulting in severity and it is documented throughout the world in all sexes, races and age group. Diseases caused by GAS ranges from mild upper respiratory tract- pharyngitis or impetigo but in extreme cases, it will lead to invasive diseases- cellulitis, bacteremia, necrotizing facilities and toxic shock syndrome (TSS).

In bacteria cell envelope, it is deemed to be great importance as the vital point for interaction between the bacterium and it environment. Bacterial cell envelope proteins carry out variety of important functions such as adhesion, nutrient acquisition and numerous amount of interaction with the host defences(Sutcliffe and Harrington 2002).

In Gram-positive bacteria, it lacks a retentive outer membrane and thus, it had evolved a mechanism for retaining proteins within their cell envelopes, including covalent linkage to the peptidoglycan and non-covalent binding to teichoic acids and other cell polymers. -

These bacterial lipoproteins have their N-terminal modified with N-Acyl Diacyl Glyceryl Group and in another possible further step whereby an additional fatty acid is amide linked to the free amino terminus which does not appear to occur in the low G+C gram positive bacteria (Lain C. Sutcliffe and Dean J Harrington, 2002).

Bacteria lipid modification enables it to efficiently carry out their important functions between the cell wall and the environment (Braun et al, 1993). Bacterial lipid modification is vital for its viability and it is ubiquitous in and unique to bacteria.

Bacterial Lipoproteins

Bacterial cell envelope proteins carry out numerous amounts of important functions between the bacterium and the environment, mainly the interaction between the host and host pathogen.

In Gram Positive due to lack of outer retentive membrane, they have established their own specialized mechanisms for retaining their own lipoprotein in their outer membrane. Among the mechanisms established in Gram positive bacterium, membrane anchoring of bacterial lipoproteins (Lpps) by covalent N-terminal lipidation is significant (Braun & Wu, 1994; Sutcliffe & Russell, 1995).

Lipoproteins lipid modification was attained through by covalently adding a diacyglycerol to the invariant cysteine residue in the lipoprotein signal peptide as was described in the Braun's lipoprotein in Escherichia coli (Braun and Wu, 1994).

Lpps performs wide range of critical functions such as substrate binding proteins (SBPs) in ABC transporter system; in antibiotic resistance; in cell signalling; in protein export and folding; in sporulation and germination; in conjugation and various other functions (Sutcliffe & Russell, 1995).

Thus, the lipoproteins of Gram-positive bacteria have been proposed to be functionally equivalent as that of Gram-negative bacteria. It was directly compared and confirmed by the fact that SBPs of ATP binding are typically lipoprotein (Nielsen and Lampen, 1982).

Translocation across cellular membrane

In order to reach their site of function, vast proportion of newly synthesised proteins need to translocate to outer cell memebrane. This is done mainly via the general secretory (Sec) pathway and Tat (twin arginine protein transport) system. The difference between the two systems lies in the protein conformation either folded or unfolded.

Prokaryotes proteins targeted to the periplasm and to outer cell membrane are usually transported across cytoplasmic membrane by the Sec pathway (Pugsley, 1993). Thus, the Sec pathway is the predominant route of transportation of proteins across the cytoplasmic membrane among the two distinct pathways (Driessen and Nouwen, 2008). In Sec pathway, the proteins with unfolded conformation have to synthesised with N-terminal sequence which will excised at a later stage during the exportation via a signal peptidase situated on the periplasmic face of the membrane. There has been findings in which some of the putative lipoproteins are exported via SecA2 accessory pathway across the cytoplasmic membrane in unfolded state (Braun and Wu, 1944 ; Froderberg, 2004).

In Tat system, proteins with folded conformation, even oligomeric proteins are transported to periplasmic membrane. Under the Tat system, protein signal sequence unusually long contains consensuses S/T-R-R-x-F-L-K in which arginine residues is invariant and often bind to redox cofactors on the cytosol (Berks, 1996; Santini et al., 1998). Lipoproteins precursor s exported through Tat system in fully folded conformation were confirmed during an analysis of dimethyl sulphoxide (Dms) reductase in Gram-negative bacteria (Gralnick, J.A. et al).

This indicates that Tat system is a mechanism which is fundamentally different from the Sec pathway and requires the proteins to be folded before they cross the cellular membrane.

Proteins destined for lipidation contains a motif in their signal peptide, lipobox which forwards them to lipoprotein biogenesis machinery after exportation via either Sec pathway or Tat system. Studies primarily based on E.coli, indicates that all newly synthesized lipoproteins are transported via Sec pathway in an unfolded conformation across the cytoplasmic membrane (Braun and Wu, 1994; Froderberg, 2004).

Lipoprotein Biogenesis

To attain their full fledged function, the newly synthesised protein exported across the cytoplasmic membrane via the Sec pathway or Tat system will be channelled to the lipoprotein biogenesis machinery. This channelling process will be guided by the conserved motif (lipobox) in the proteins signal peptides to the machinery. Lipoprotein biogenesis pathway was well characterized in the studies of E.coli (Braun and Wu, 1994).

Figure 2: Type I and Type II signal peptides via Sec and Tat Dependent Transport (a) General Secretory Pathway, (b) Tat System

Type I and Type II signal peptides composed of three distinct segments, a positively charged amino-terminal N, a central, H-(hydrophobic) region and a more polar carboxy terminal C- (cleavage region) (von Heijne,1985).Within type I signal peptide, it sustains a recognition motif (A-X-A, where X can be any amino acid). In the case of Type II signal peptide, it contains the recognition motif sequence of (L-3-[A/S/T]-2-[G/A]--1-C+1) and its cleavage site often referred as lipoprotein 'lipobox'. It is distinct in its way that it has conserved SRRXFLK sequence between N-region and H-region (Petit, C.M et al), within which the twin arginine (RR) motif is almost absolutely conserved. The common residue among all the bacterial lipoproteins is the conserved cysteine +1 of the Type 2 signal peptide lipobox (L-3-[A/S/T]-2-[G/A]--1-C+1).


Figure 2: Biogenesis of Lipoproteins

Bacterial lipoprotein biosynthesis characterization in E.coli done via a distinct and conserved pathway is unique to prokaryotes (Braun, V., and H.C.Wu, 1993).Once the protein is exported across the cellular membrane with the guidance of the signal peptides via Sec or Tat dependent system, the conserved cysteine residue within the lipobox of the signal peptide is modified with a diacyglycerol group attached through a thioether linkage. The above whole reaction is catalyzed by the enzyme prolipoprotein diacylglycerol transferase (Lgt), using phospholipid substrates (Sankaran et al., 1995; Qi et al., 1995).

Once after the conserved residue gets lipdated, the signal peptide is cleaved within the lipobox by a specific lipoprotein signal peptidase II (Lsp), enzyme to release the lipidated cysteine as the N-terminal for the mature bacterial lipoprotein (Braun & Wu, 1994; Sankaran & Wu, 1995). The signal peptidase II requires diacylglycerol modification prior to cleavage and this identifies that diacylglycerol modification must done before the cleavage process by Lsp, enzyme (Hussain, M et al, 1980; Dev, I.K. and Ray, P.H .1984). Above mentioned steps for lipidation of Gram-positive bacteria, it was confirmed to be vital and sufficient.

In the third step modification of the lipoprotein in the biogenesis pathway would be the lipid modification of the lipoprotein by a fatty acylation of the amino group of the N-terminal diacylglycerol modified Cys to form N-acyl diacylglycerol Cysteine by the enzyme, lipoprotein amino acyl transferase. This enzyme is not conserved as its homologues are not available in the genomes of low G-C Gram positive bacteria (Tjalsma et al., 1999 and our unpublished observations).

In summary in order to attain a full functional mature lipoprotein, it is dependent on the efficient function of the signal peptide and the lipobox within it. The signal peptides were well established as they consist of three different regions: N-domain, containing a positively charged amino acids lysine and / or arginine, central hydrophobic H-region and a C-region (cleavage site for signal peptide II). These regions must be fully functional to export the premature lipoprotein across the cellular membrane. The lipobox within the lipoprotein has to be place appropriately for recognition of prolipoprotein and modification by Lgt and Lsp enzymes to attain a mature lipoprotein (Klein et al., 1988; von Heijne, 1989; Braun & Wu, 1994; Tjalsma et al., 2000).

Bioinformatic Prediction of Lipoprotein in Gram-positive Bacteria

With the well established features of signal peptide, bioinformatic analysis of these signal peptides will be able to predict the number of lipoproteins in Gram-positive bacteria using the signal peptide sequence features and the well conserved lipobox sequence within it.

Signal peptides tend to be shorter that secretory signal peptides which indicate that the c-region is shorter and contains apolar amino acids when both Gram-positive and Gram-negative signal peptides were analysed. It implies that it is a continuation of the hydrophobic domain which is differentiated primarily by the sequence conservation preceding the invariant lipid-modified cysteine.

It is this sequence, typically Leucine−3-Alanine/Serine−2-Glycine/Alanine−1-Cysteine+1 at positions -3to +1 that is referred to as the lipobox sequence and the consensus pattern of amino acid usage have been documented in Prosite under the pattern entry PS00013/PDOC00013. Based on these sequences, there were several pattern expressions being modified to reduce false positive lipoprotein being predicted in the Gram-positive genome wide search.

In the initial Prosite consensus pattern syntax PS0013 (the sequence motif determining lipidation), the permitted lipobox amino acids preceding the cysteine at position-1 to-4 have been indicated and{DERK}(6)-[LIVMFWSTAG](2)-[LIVMFYSTAGCQ]-[AGS]-C wherein the permitted lipobox amino acids preceding the cysteine at positions -1to -4 are indicated and the absence of charged residues (i.e. no D, E, R or K) within the signal peptide h-region is prescribed. Furthermore, the pattern is subject to the application of additional rules: Firstly, the cysteine must be between positions 15 and 35 of the sequence under consideration and secondly, there must be at least one lysine or one arginine in the first seven positions of the sequence. These rules localize the pattern to N-terminal sequences with n-regions characteristic of signal peptides. Membrane spanning domain (MSD) in protein sequence was also predicted setting of 14 aa for hydrophobic domain.

Large number of putative genes was identified using the N-terminal sequence of the signal peptide in various Gram-positive bacterial genomes. For determining the false-positive bacterial lipoproteins, they were analysed individually, searching the N-terminal MSD and also the additional number of MSD using TMpred. In Bacterial Lipoprotein sequence in which there is absence of MSD or there is extension of N-terminal MSD beyond the cysteine lipobox were considered possible false-positive. Other sequence analysis was done by SignalP as the MSD analysis was not sufficient for justification for false positive lipoprotein as both the Ctac and the QoxA proven Lpps had two additional MSD predicted beyond their N-terminal lipid anchors. For this sequence analysis conditions that were considered to prove Lpps were false-positive were signal peptide features were absent altogether and /or the lipobox cysteine was internalised rather than to terminal to n h-region/MSD. For sequences which require further clarification, they were analysed using (notably TopPred2 and DAS) predicting transmembrane regions and a consensus taken as to the position of the putative lipobox cysteine relative to the length of the first predicted MSD.

Signal peptide features can be described using 'pattern expressions' written in Prosite syntax as shown in Table I. These patterns can be used for the bioinformatic identification of bacterial lipoproteins. '<' indicates the pattern is restricted to the N-terminus of the protein and at each position thereafter the amino acids shown are either permitted (square brackets) or prohibited (curly brackets). X is any amino acid. Where stretches of amino acids can vary in length, the range is indicated in parentheses. The original G+LPP pattern was described by analysis of the signal peptide features of 33 experimentally verified lipoproteins [54]. An extended dataset of 90 experimentally verified lipoprotein signal peptides was used to revise this pattern (G+LPPv2; [55]). The essential cysteine is considered the +1 position and, along with amino acids at positions

-3 to -1, constitutes the 'lipobox'.

The Prosite profile P51257 (originally pattern PS00013) is based on the analysis of signal peptides from Gram-negative and other bacteria [53] and is notably more relaxed in the -2 and -3 positions.

Based on these sequences, there were several pattern expressions being modified to reduce false positive lipoprotein being predicted in the genome wide search.

To derive to the prediction of lipoproteins using bioinformatic analysis, Sutcliffe and Harrington, 2002 in their experiment had created a dataset of experimentally verified Gram-positive Lipoproteins that have lipidation and performed bioinformatic analysis to create a revised pattern for identify Gram-positive bacterial putative lipoproteins (Sutcliffe and Harrington 2002).There were several requirements were considered to prove the lipoprotein is lipidated. They were (1) metabolic labelling of the protein, in its source organism, with exogenously supplied radiolabelled fatty acid (normally palmitate, incorporation of which is typically detected by autoradiography following protein electrophoresis). 2) Inhibition of Lpp signal peptide processing by treatment with the antibiotic globomycin, which specifically inhibits Lsp (Inukai et al., 1978). (3) Chemical characterization of the purified protein. (4) Evidence that protein processing is disrupted by mutation in either Lgt or Lsp, or following site directed mutagenesis to replace the lipobox cysteine. Within this set of criteria and along with extensive review of scientific journals, 33 proteins were identified as proven bacterial lipoprotein and were tabulated below in the figure. Streptococcus Pyogenes LppC protein was identified with inhibition of Lpp signal peptide processing by treatment with the antibiotic globomycin, which specifically inhibits Lsp (experimental evidence 2).