Staphylococcal Virulence Factors Synthesis - Assignment Example

Staphylococcal Virulence Factors Synthesis al Affiliation Staphylococcal Virulence Factors Synthesis When you compare the phenotype of arg+ to arg- mutants, you note that arg+ strains can synthesize a number of secreted proteins like serine protease, hemolysins, and toxic shock syndrome toxin-1 (TSST-1). These chemical substances are synthesized at very low levels or not at all in arg-. Surface proteins such as coagulase, fibronectin binding protein, and protein A are produced in arg- but at a lower level than in arg+ (Novick, Ross, Projan, Kornblum, Kreiswirth & Moghazeh, 1993). Cloning and subsequent sequencing of the arg locus proved the existence of two very different operons, P2 and P3. P3 initiates RNAIII that codes 6-hemolysin. P2 operon is 3Kb in length. It is unique when compared to P3 since it has up to four open reading frames, agrA, agrB, agrC, and agrD. The agrA controls the sensory transduction while agrB is responsible for the production of histidine phosphokinase in bacteria. Due to mutations, defects may occur in any one of four open reading frames, the resulting strains don’t have P2 and P3 transcripts, beside they become arg-. These strains cannot initiate transcription from the P2 and P3. Insertion at C1Ai site in the RNARIII region in P3 results in inactivation of arg functions. 2. a) β-lactamase has higher activity as compared to the normal P2 and P3 promoters, and thus β-lactamase can make other existing non-useful plasmids like p1524 unstable. Using P2 and P3 instead of β-lactamase allows expression of the p15424, and this interferes with the results. b) RN6390B, arg+ produces stronger signals for alpha and beta hemolysins than does RN69 11, a null-arg strain, whose signal is extremely weak. If the researcher used arg+ instead, he would not obtain the same results due to interference by other unnecessary signals. c) When a B lymphocyte secretes antibodies that are directed towards a specific epitope on an antigen, we call the antibodies monoclonal. However, when a significant number of antibodies are secreted that have different affinities and specificities towards different epitopes, we call them polyclonal antibodies. B- hemolysin antibody is a polyclonal antibody, has a wide range of affinities and specificities and thus it can detect any antigen present in its environment. d) In anti-b blot, protein A produces a stronger signal for arg-a42 and no signal in arg-a40. The signal for B-hemolysin is present only in arg-a40 and not arg-a42. These results mean that different regions of P3 codes for different chemicals. 3. The author used RN7220 because it can increase the hla (a- hemolysin coding sequence) and hlb (B-hemolysin coding sequence) transcripts while suppressing spa transcripts. RN7220 has RNAIII clone a27 (pRN6735) as its genotype. From the fusion experiments, the researchers learned that B-lactamase expression is controlled by arg since fusion produced B-lactamase expression only in arg+ strains but not arg- strains. Besides, the results also proved that arg does not have any effect on the mRNA stability but it acts through regulation of the transcription process. 4. a) Transcription fusion is a technique that aims at measuring the transcription activity of the promoter by tagging. While, translation fusion allows to you follow up, locate and note the expression of a tagged protein. b) The hollow ones represent RN6390B strain, and the filled ones represent ISP546. It is because we noted earlier on that the a27 clone promotes hla transcripts, and it decreases the spa transcript in arg+. From our graphs, the hollow ones have increased more than the filled ones in a-hemolysin and vice versa in protein A. c) The conclusion was reached at due to the study of subclones in which some regions of RNAIII have been deleted and in some, the reading frames were inactivated by mutagenesis of specific sites on RNAIII. It was proven that despite the deletions the 3subclones like a39 and a28 still activate hla and hlb transcription more strongly to get to maximum activity at a length of 422 nt. Besides the 5 subclones also caused hla and hlb transcription. RNAIII regulate hla translation and transcription independently. 3 subclones like a28, a39, and a41, cause expression of hlb but not hla. It shows that hla mRNA translation does not take place in these strains with 3 subclones. However a-hemolysin expresses itself in strains with 5 subclone, a29. It shows that the translation of mRNA cannot happen in absence 5 region of RNAHI. 5. a) The mechanism of actions proposed was that, RNAIII is the effector by itself. Secondly, the translation products may act as effectors and thirdly open reading frames under the influence RNAIII, act via the products of translation or by the effects of open reading frame translation on RNAIII secondary structure. b) The author constructed and analyzed 5 and 3 subclones, in which some regions of RNAIII have been deleted. Besides some reading frames were also inactivated using site-specific mutageneses. Site-directed mutagenesis included deleting 13 amino acids or introduction of nonsense mutation at hld codon 3. The researchers measured the regulation by noting how weak or strong the inducement was, for example, a39 and a40 induce hla and hlb more strongly than the a41. The spa repression in 5 was weaker when compared to 3 subclones. c) From experiments with sheep blood agar and Western immunoblotting, it is noted that a28, a39, and a41, failed to stimulate a-hemolysin but stimulated B-hemolysin translation. Besides, when the cytoplasms of the respective clones were studied, there was no a-hemolysin found. It may be attributed to degradation of the hemolysin. However, degradation was ruled out following a study that showed that equal levels of a-hemolysin antigen exists in both wild-type strain and arg-a41 strains. We can conclude that 5 subclone and the 95 nt region of 3 subclone are essential for hla translation, and that hla translation and transcription is regulated independently. 6. a) It proves that the hla translation does not depend on its transcription. Transcription and translation are two independent processes. b) 5 subclone is essential for hla translation. However 5’ end of the leader of the hla mRNA cannot act alone, it needs the presence of 5’ end of RNA III. Messenger RNA passes on genetic information, which means there has to be the recipient f that information before the translation is induced. 7. As noted earlier β-lactamase has higher activity as compared to the normal P2 and P3 promoters. When B-lactamase promoter is used to induce RNAIII, it restores the negative and positive regulation of the arg system even when the chromosomal locus in the host strain has been deleted. It means that the strain will produce the secreted proteins like serine protease, alpha, beta and gamma hemolysins. Besides, surface proteins such as coagulase and protein A will be synthesized and expressed. The regulatory functions of these proteins are vital to the survival of the microorganism and despite the external inducement, the secretion and expression of this exoproteins especially in arg- afford the required regulation. 8. The author proposes that RNAIII may control transcription by controlling the synthesis of accessory protein factors or through the formation of stable complexes with the factors. RNA-protein complex existence has been proved by reacting lysates of the crude cell with SDS which resulted in a drop in the sedimentation rate of RNARI. Secondly, the author notes that up-regulation of the exoprotein genes beyond the exponential phase needs a second activating signal. Finally, drugs like erythromycin resemble RNAIII on target gene transcription where induce up-regulated genes and repress down-regulated genes. The possibility of RNAIII reacting directly with regulatory sequences in target DNA should not be overruled. 9. The P2 contains the following reading frames, agrA, agrB, agrC and agrD, the respective functions have been discussed. On the other hand, P3 initiates a 0.5 kb transcript, RNAIII. RNAIII encodes which 6-hemolysin, insertion in this region inactivates agr function, but the two promoters are not affected because the products of the four P2 open reading frames work together activate transcription From fig 1B, a hld reading frame that is translated is preceded by 18 in-frame codons (pre-hld). These codons are not translated, and the pre-hld has a plausible Shine - Dalgarno site. I think it is because of these structural differences that bring about the inactivation upon insertion. 10. a) The protein is glycosyltransferase in Enterobacter cloacae ECNIH2. b) The identity of the protein explains the identity of the strain because alongside the name of the protein, can relate to different bacteria like in my case, proteobacteria, gammaproteobacteria among others. Glycosyltransferase is an anabolic enzyme, protein in nature that is involved in the synthesis of simple and complex carbohydrates. It acts by adding monosaccharides from activated donors. References Novick, R. P., Ross, H. F., Projan, S. J., Kornblum, J., Kreiswirth, B., & Moghazeh, S. (1993). Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. The EMBO Journal, 12(10), 3967. Read More