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Prolipoprotein Diacylglyceryl Transferase (LGT) is an important enzyme which catalyzes the lipid modification of bacterial lipoproteins. The importance of this enzyme as a role in producing matured lipoprotein is widely researched. Ability of LGT is producing lipoproteins to play a role in cell-cell adhesion, protein maturation and nutrient acquisition leads to the survival and colonization of these bacteria within the host cell. In this report, the ability of LGT to cause the virulence in gram positive bacteria is reviewed. Study on the ability to cause virulence in bacteria and the cause of pathogenicity can lead to findings of cure in diseases caused by gram positive bacteria.
Bacterial lipoproteins are unique and ubiquitous to the specific bacteria. Lipoproteins have been identified to contain N-acyl Diacyl Glyceryl as their N-terminal amino acid. Tokunaga et al. proposed the first biosynthetic pathway in 1982. The study was carried out using Braun's lipoprotein of Escherichia Coli, in vitro and in vivo. Tokunaga and team demonstrated that prolipoprotein-phosphatidylglycerol glyceryl transferase catalysed the transfer of non-acylated moiety of phosphatidylglycerol (PG) to the sulfhydryl group of the prospective N-terminal cysteine residue in the unmodified prolipoprotein. The diacylglyceryl modified prolipoproteins is formed when one or more O-aryltransferase acylates the sn-3 and sn-2 hydroxyls of the glyceryl moiety of glycerylcysteine (Tokunaga et al., 1982). This pathway was modified Sankaran and Wu in 1994 with the findings that showed diacylglyceryl modification of prolipoprotein occurs when diacylglyceryl moiety from PG is transferred to the cysteine residue to form diacylglyceryl-prolipoprotein with the formation of sn-1 phosphate. The enzyme that catalyzes this reaction is prolipoprotein-phosphatidylglycerol diacylglyceyl transferase (LGT) (Sankaran and Wu, 1994). In the study carried out, lgt was shown to be one of the genes that encodes for prolipoprotein modification and enzyme processing. Thus, the conclusion derived from the study suggested that lgt gene is essential for lipid modification.
Figure 1: Prolipoprotein Diacylglyceryl Transferase (LGT) in Lipid Modification pathway (Sankaran and Wu, 1994)
This pathway is essential for bacterial growth, viability and cell division. Analysis of this enzyme provides information about the novelty of reaction which is catalyzed by LGT, its ubiquity in bacteria and importance as the first step in the lipid modification pathway. Lipoproteins are known to play a role in cell-cell adherence, protein maturation, cell signalling and also nutrient acquisition (Hamilton et al., 2000). Bacterial lipoproteins also play a role in survival of bacteria within the host cell and colonization of bacterial in the host cells (Sutcliffe and Harrington, 2002; Sutcliffe and Harrington, 2004; Sutcliffe and Russell, 1995). Thus, understanding of the lipid modification pathway and the role of LGT is important to determine the processes which are affected by LGT. Importantly, its role in cell-cell adherence plays a role in adhesion of bacteria to the cells, causing its pathogenic reaction thus leading to the disease (Hamilton et al., 2000)..
According to MeSH, virulence is the degree of pathogenicity within a group of microorganism as indicated by the fatality rates or the invasion rate of microorganism into the tissues of host. This pathogenicity capability is determined by factors causing its invasion or fatality.
Comparison with gram negative bacteria
Relationship between the structure and function of Lipoprotein Diacylglyceryl Transferase was studied by comparing its effect in different bacteria and effects of mutant LGT in bacteria. To study the relationship between the structure and function of LGT, Qi et al., 1995, carried out a research using a clone containing LGT gene, lgt, which was isolated from Staphylococcus aureus to study its effect in the bacteria.
Amino acid comparison of LGT from Staphylococcus aureus with LGT sequences of gram negative bacteria from the GenBank database was carried out. A high level of homology was found with LGT of Escherichia coli, Haemophilus influenzae and Salmonella thyphimurium which were previously cloned. A stretch of hydrophobic segments interrupted by hydrophilic segments were significantly noticed. The observance of 24% identity and 47% similarity shows that S.aureus is not 100% similar to the gram negative bacteria. This shows that the bacteria differs in the lipid modification process as well, indicating the effect of LGT on both type of bacteria can be different.
Solid boxes represent identity
Dashed boxes represent similarity
. . . . . . represent the gap in the sequence
Figure 2: Amino acid sequences of LGT from E.coli, S.typhimurium, H.influenzae and S.aureus. Amino acid comparison of these sequences showed a 24% identity and 47% similarity.
At neutral pH, S.aureus has a net charge of +9. This is due to the abundance of Arginine and Lysine amino acid in about equal amounts. The other three gram negative bacteria revealed predominant levels of Arginine and a few Lysine residues. Based from the comparison of net charges, LGT of S.aureus is the highest compared to those of the gram negative bacteria.
Net Charge of LGT
Table 1: Net charge of LGT of gram positive and gram negative bacteria at neutral pH
The pI level of LGT in S.aureus was around 10.40. N-terminal and the middle region of LGT were observed to be more conserved than the C-terminal region in S.aureus. Codon usage of S.aureus lgt gene differs from the codon usage of gram negative bacteria. The lgt gene organization in S.aureus differs from the ones in gram positive bacteria. In S.aureus, thyA gene is not next to the lgt gene. A significant stretch (H-103-GGLIG-108) was identified in the S.aureus LGT enzyme. The stretch contains an invariant Histidine (His-103) which shows the possible association with the calalytic activity of the LGT enzyme. The size of LGT in S.aureus is smaller than the LGT in E.coli and S.typhi by 12 amino acids. It is however larger than the LGT in H.influenzae by 11 amino acid. Based on Chou-Fasman prediction, LGT in S.aureus is high in Î±-helix-breaking amino acid, glycine and proline. Staph aureus also has predominant levels of Î²-sheet structure and low levels of Î±-helix. This is however similar to the other gram negative bacteria used in this study.
Aim of Study
In this report, the analysis of LGT as a virulence factor will be studied particularly in gram positive bacteria. To study the effect of LGT on the virulence of gram positive bacteria, analysis was done of several gram positive bacteria. Below is the list of gram positive bacteria which was reviewed in this report:
Gram positive bacteria
Table 2: List of gram positive bacteria reviewed in the report
Study of LGT in Staphylococcus aureus
Staphylococcus aureus is known to cause diseases such as pneumonia, bacteremia,osteomylitis and other skin infections. These diseases occur when Staphylococcus aureus overcomes the physical barrier and invades the tissues via contaminated medical devices or surgical wounds (Lowy, 1998). Immune system responses towards the pathogen, Staphylococcus aureus, when it invades the cells. Bacterial lipoprotein which functions as pathogen-associated molecule patterns (PAMPs) bind to the Toll-like receptors (TLRs) which triggers a signaling cascade in response to the pathogens (Aliprantis et al., 1999; Brightbill et al., 1999; Hirschfeld et al., 1999; Medzhitov et al., 1997; Medzhitov, 2001 and Yoshimura et al., 1999). In short, these bacterial lipoproteins trigger an immune response in case of a Staphylococcus aureus infection (Stoll et al., 2005). Bacterial lipoproteins have been observed to Aliprantis et al., 1999; Brightbill et al., 1999; Hirschfeld et al., 1999; Norgard et al., 1996 and Rawadi et al., 1999). Stoll and team investigated the contribution of LGT towards the virulence of S.aureus by observing the immune response of lgt mutant in the host cell (Stoll et al., 2005). In the study carried out, a S.aureus lgt::ermB mutant was constructed by replacing lgt gene with an erythromycin resistant cassette (ermB). As lgt gene was replaced, lipid modification would not be able to take place and its effects on invasion into the host cells and cytotoxic effects were observed. One of the observations from the study includes the amount of lipoprotein detected. Most lipoproteins were found in the membrane, low levels in the cell wall and culture environment in which the bacteria is kept. This surface bound and lipoproteins which are released into the environment can interact with the host immune system (Stoll et al., 2005). Epithelial cells which acts as barrier against pathogens and activate phagocytic cells by releasing chemokines and cytokines. A549 pulmonary type II epithelial cells were used to determine the effect of inflammatory immune response towards lgt mutant. These epithelial cells were infected with both lgt mutant S.aureus and wild-type S.aureus (lgt gene functions as normal). Induction of IL-6, an inflammation activator, and IL-8, a neutrophil activator, was assessed. More cytokines were observed in the presence of lgt gene compared to the cells with mutant lgt gene. This shows that lipoproteins present in the cells are the cause of cytokine and chemokine induction. Stoll and team also reported the absence of LGT in the host cells fails to induce Tumour Necrosis Factor-Î± (TNF-Î±) response (Stoll et al., 2005). Missiakas and team also demonstrated that mice infected with lgt mutant bacteria die rapidly compared to mice with an lgt insertion (Missiakas et al., 2006). Lipid modification plays a role in inducing and immune response against S.aureus and LGT being the first step in this process, definitely plays an important role in inducing a response in the virulence of this gram positive bacteria.
Study of LGT in Listeria monocytogenes
Listeria monocytogenes,a facultative intracellular pathogen is known to cause food-borne disease in both humans and animals (Seeliger, 1988). Listeriosis, as the disease is named leads to syndromes such has meningitis, encephalitis, abortion and sepsis. Lgt, the key enzyme involved in lipoprotein processing plays a role in the virulence of L.monocytogenes. PrfA, a positive transcription regulator, controls the virulence factors of Listeria monocytogenes (Baumgartner et al., 2007). This enables the bacteria to invade and multiply in cells despite the barriers present in mammals. The absence of Lgt impairs growth of L.monocytogenes intracellularly. This causes the release of lipoproteins in to the culture supernatant. The high levels of lipoproteins in the supernatant enable the identification of PrfA-dependent lipoproteins. Over expression of PrfA increases the virulence factors of L.monocytogenes as observed in this study carried out. High level of conservation is observed for Lgt in Listeria, showing a 98 to 100% identity. In the study carried out by Baumgarter M. et al showed that the deletion of lgt gene which encodes the Lgt enzyme, in L.monocytogenes leads to defect in the bacterial cell growth intracellularly and higher levels of lipoprotein was detected in the culture environment. This strengthens the fact that Lgt is needed for full virulence of L.monocytogenes. Absence of Lgt results in the defect in bacterial cells growth which leads to inability of the bacteria to carry out its function. Lgt gene was deleted by constructing an in-frame deletion. This mutant strain was similar to the original strain in cell and colony morphology. Baumgartner et al demonstrated the gene deletion of lgt does not affect cell growth in the brain heart infusion medium, a medium enabling bacterial cell growth as the growth analysis showed similarity of growth rates at the logarithmic phase. However, in stressful conditions such as lack of nutrients, during cell invasion or intracellular growth, mutant lgt strain seems to have a reduced growth compared to the wild type strain. Infection of both wild type and mutant lgt strain on the mouse fibroblast cell line showed the contribution of lipidation to the pathogenicity of Listeria monocytogenes. This again shows the effect of LGT on the ability of Listeria monocytogenes to cause the disease. Results indicated that lgt did not interfere with movement of the bacteria within the cells. Lgt deletion also did not affect the entry of these gram positive bacteria into the mammalian cell lines. Interestingly, hours after the infection of mutant lgt, there were low levels of this mutant lgt in the mouse cell line. This strengthens the observation that Lgt plays a role in intracellular growth in L.monocytogenes (Baumgartner et al., 2007). This lgt gene deletion in L.monocytogenes showed the contribution of Lgt towards the virulence of the bacteria. Lgt is one of the virulence factors in this bacteria but it has to be noted that Lgt alone is not the cause of this bacteria's pathogenecity. Another study showed that L.monocytogenes which are depriveded of lgt gene are not able to induce NF-ÎºB activation via TLR2 induction. Lipoproteins were shown to be ligands of TLR2, causing to play an important role in bacteria recognition during an infection by L.monocytogenes (Machata et al., 2008).
Study of LGT in Streptococcus agalactiae
Gram positive bacteria, Steptococcus agalactiae is categorized under the Group B Streptococcus (GBS). S.agalatiae is the leading cause sepsis and meningitis in newborns (Fluegge et al., 2005 and Luck et al., 2003). The infection of this gram positive bacterium leads to highly inflammatory diseases with a death rate of approximately 10%. A series of Nitric Oxide (NO), cytokines and intracellular signaling intermediates is release by GBS when the host in infected in vitro (Henneke & Berner, 2006). TLR2, known as a signaling molecule was found to be essential in a mouse model which was infected with GBS sepsis (Mancuso et al., 2004). GBS interaction with TLR2 is helpful when in low dosage but becomes fatal in higher dosage (Henneke et al., 2005). Henneke et al., 2005 carried out a research aiming to identify the inflammatory molecules released by GBS which stimulates TLR2. GBS mutant which was deficient in Bacterial Lipoproteins (BLPs) were found to be impaired in activation of TLR2. N-terminal acylation of prelipoproteins which were mediated by LGT was essential to the released factors of GBS to activate TLR2, both in vitro and in vivo. LGT is among the enzymes which cause maturation of BLPs by catalyzing the acylation of signal peptide lipobox. In the experiment carried out by Henneke and team, lgt gene was inactivated in a GBS strain which was isolated from an infant diagnosed with fatal septicemia. The role of protein acylation was studied in this inflammatory potency of GBS. Comparison was done between both wild-type strain and mutant with an inactivated lgt gene. The GBS was cultured in the presence of [2H] palmitate to insert the labeled acyl anchors into the N-terminus of lipoproteins. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) was carried out to separate the bacterial cell extracts. Many bands were observed in the wild-type strain of GBS and no labeled bands were detected in the lgt mutant strain of GBS, confirming both the presence and absence of lgt in wild-type and mutated strain individually. Cell culture supernatant with the mutated lgt GBS strain showed low inflammatory activation of RAW macrophages. This was assessed by the release of TNF (Figure 3). Wild-type strain with the presence of the lipoprotein lead to the NF-ÎºB activation in a TLR2 assay.
Figure 3: Analysis for TNF induction in RAW macrophages in the wild-type strain
Figure 4: Analysis for TNF induction in RAW macrophages in the mutated lgt strain
Cytokine formation was observed in macrophages, stimulated by GBS with lgt strain (Figure 4). Thus, maturation of BLPs affect the TLR2 activation. Interaction of GBS and TLR2 is not critical for cytokine induction in macrophages. However, it is important to note this interaction at the cell-cell interface. Protein acylation is an important process required for the interaction of GBS-BLPs and TLR2. This protein acylation is carried out by LGT, the enzyme studied in this research. In the presence of BLPs, GBS containing lipoteichoic acid (LTA) activates TLR2. LTA is known as an important TLR2 agonist which binds to TLR2 and triggers its response (26-28). LTA from lgt mutant showed little effect compared to the LTA from the wild-type strain. BLP, a GBS product, interaction with TLR2 which contributed to the GBS sepsis in mice is an early recognition of GBS. This mechanism alerts the immune system during GBS sepsis. If there is insufficient interaction between BLPs and TLR2, occurance of inflammation, organ failure or even death can be observed (Henneke et al, 2008). In a separate study conducted, mutated LGT enzyme did not affect cell viability of S.agalactiae, however, pleiotropic effects due to the mutated enzyme is significant to the virulence of this gram positive bacteria (Bray et al., 2009).
Study of LGT in other gram positive bacteria
Streptococcus equi is known to be the cause of strangles (Timoney, 2004); a pharyngeal constriction is the upper respiratory tract of a horse (Slater, 2003). Hamilton and team concluded that lipid modification pathway is the cause of S.equi pathogenecity. Study was carried out in both mouse models and pony models. Failure of mice to gain weight and early signs of strangles disease such as nasal discharges, swelling of lymph nodes and pyrexia in ponies confirmed the findings that presence of LGT is one of the virulence factors in S.equi (Hamilton et al., 2006).
Viridan group of streptococci consist of Steptococcus sanguinis (Carlsson, 1965). S.sanguinis causes endocarditis, an infection of the valves or lining of the heart (Moreillon and Que, 2004). Rheumatic fever occurance causes serious damage to the heart which leads to endocarditis (Moreillon and Que, 2004; Wilson et al., 2007). Mutated lgt gene used in this study of S.sanguinis indicated a loss of protein labelling (Das et al., 2009). This indicated a lack of acylation (Lai et al., 1980) causing a defect in the virulence of S.sanguinis. The overall picture of this study indicated that LGT affects the virulence of this gram positive bacterium. Mutated form of this enzyme indicated the reduced virulence in S.sanguinis (Das et al, 2009).
Prolipoprotein Diacylglyceryl Transferase is an enzyme which plays an important role in the first step of the lipid modification pathway (Sankaran and Wu, 1994). Lipoprotein, catalyzed by LGT, plays an important role in cell-cell adhesion (Hamilton et al., 2000). Cell-cell adhesion is important in bacterial adherence towards the host cells. Adherence to the host cells will enable bacteria to pass on the virulence factors to the bacteria. Failure in LGT enzyme to function can result in release of lipoproteins into the culture medium. This may cause virulence causing proteins to be degraded (Hamilton et al., 2006). Absence of lipid modification does not affect the cell viability of L.monocytogenes (Baumgartner et al, 2007). This supports the data shown for all S.aureus, B. subtilis and S.pneumoniae (Leskela et al., 1999; Petit et al., 2001 and Stoll et al., 2005). Defect in LGT can affect virulence in the gram positive bacteria but cell count of bacteria is not affected as bacterial growth continues. In study done using S.aureus model, lack of lipid modification played a role in the immune response (Stoll et al., 2005). This indicated that defect in LGT, leading to lack of lipid modification, affecting the host's immune response towards the bacteria. Loss of surface lipoproteins to play a role in cell-cell adhesion reduces recognition by TLR2. This causes enhanced virulence in S.aureus despite the mutated lgt gene (Stoll et al., 2005; Wardenburg et al., 2005). However, Streptococcus pneumoniae was shown to not exhibit much virulence in the presence of a mutated lgt gene (Khandavilli et al., 2008). Absence of LGT is able to impair growth of gram positive bacteria especially L.monocytogenes intracellularly. This will cause the release of unmodified lipoproteins into the culture supernatant enabling identification of PrfA-dependent lipoproteins. Bacteria will be able to invade and multiply in the host cell by the identification of PrfA as it controls the virulence factors of L.monocytogenes. (Baumgartner et al., 2007). Baumgartner et al., 2007 also showed that effect of cell growth intracellularly leads to the release of lipoproteins into the cell environment, thus, reducing the full virulence of L.monocytogenes. TLR2 induces NF-ÎºB activation. However, reduced or impaired lgt gene is unable to activate TLR2 as shown in the study of L.monocytogenes (Machata et al., 2008) leading to impaired immune response towards the gram positive bacterial infection. Lipoprotein, being one of the important parts of an ABC transporter system is involved in nutrients uptake by the gram positive bacteria, bacterial stress response, and many of which is important for bacterial growth and survival. Unmodified or unprocessed bacterial lipoproteins can lead to an impaired ABC transporter system. This shows the significance of ABC transporter which contains lipoprotein to play a role in the virulence of gram positive bacteria (Polissi et al., 1998). Protein acylation was noted to be an important event in the GBS-BLPs interaction with TLR2 as observed in the study carried out in Group B Streptococcus, S.agalactiae (Henneke et al., 2008). As protein acylation is carried out by LGT, impaired LGT can cause reduced interaction with TLR2, leading to impaired virulence of S.agalactiae. To summarize, LGT play a role in acquiring a matured lipoprotein in gram positive bacteria. Matured lipoprotein is able to cause full virulence in gram positive bacteria. Absence of this enzyme can result in impaired virulence. Ability to carry out the pathogenic functions as a bacteria will be impaired. Thus, it is important to understand the role of LGT which synthesizes lipoproteins in order to deduce therapies for bacterial infections.
LGT is an essential enzyme in gram positive bacteria as it catalyzes the first step in the lipid modification pathway giving rise to matured lipoproteins. Based on the journal reviews on gram positive bacteria, LGT was shown to play a role in the virulence, degree of pathogenecitiy, in the bacteria. Absence of lgt gene showed low levels of bacterial pathogenicity, the ability of bacteria to cause diseases.
Further research has to be done to observe if S.aureus lgt mutant shows attenuated virulence in vivo (Stoll et al., 2005). Further study on bacterial lipoproteins to be used as vaccines can be done as bacterial lipoproteins are highly accessible to the host cells on the surface of the bacteria (Lei et al., 2004 and Tettelin et al., 2002).