Whole Genome Sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection

1. Introduce us Read titleIntroduce us Read title

2. Introduce PNAS High impact factorIntroduce PNAS High impact factor

3. We analysed this article, published in PNAS vol 102, n�37, September 13, 2005 It is a very recent articleWe analysed this article, published in PNAS vol 102, n�37, September 13, 2005 It is a very recent article

4. Outline Brief introduction Genome features Comparative genomics Results and discussion Summary This will be the structure of our reportThis will be the structure of our report

5. Staphylococcus saprophyticus is a Gram-posite uropathogenic of uncomplicated urinary tract infection bacteria Facultative anaerobic, cocci form and forms irregular clusters Coagulase nagative staphylococciStaphylococcus saprophyticus is a Gram-posite uropathogenic of uncomplicated urinary tract infection bacteria Facultative anaerobic, cocci form and forms irregular clusters Coagulase nagative staphylococci

6. This bacteria is known for it�s uropathogenicity and frequently causes urinary tract infection in young and middle aged female outpatients rather than hospitalized patients with indwelling cathetersThis bacteria is known for it�s uropathogenicity and frequently causes urinary tract infection in young and middle aged female outpatients rather than hospitalized patients with indwelling catheters

7. Exploring the genome atlas database, we can find useful information regarding this bacteria�s genomeExploring the genome atlas database, we can find useful information regarding this bacteria�s genome

8. The sequenced Staphylococcus saprophyticus type strain was the ATCC 15305 one We can see that this type strain contains a circular chromossome composed of 2 and a half million base pairs and 2,446 predicted ORF�s and 2 plasmids with 38,4kbp and 28,8kbp AT content is fairly high and very similar to other sequenced staphilococci genomes, as we will see further in this presentationThe sequenced Staphylococcus saprophyticus type strain was the ATCC 15305 one We can see that this type strain contains a circular chromossome composed of 2 and a half million base pairs and 2,446 predicted ORF�s and 2 plasmids with 38,4kbp and 28,8kbp AT content is fairly high and very similar to other sequenced staphilococci genomes, as we will see further in this presentation

9. The DNA Structural Atlas is a method of visualising structural features within large regions of DNA. The blue areas denote some areas corresponding to highly expressed genes ( lower intrinsinc curvature, stacking energy, position preference and AT content). These areas have also a greater stability, and usually code to rRNA molecules The region denoted by the circle corresponds to an area near the origin with lower curvature, stacking energy and position preference right before another area with greater flexibility. The DNA Structural Atlas is a method of visualising structural features within large regions of DNA. The blue areas denote some areas corresponding to highly expressed genes ( lower intrinsinc curvature, stacking energy, position preference and AT content). These areas have also a greater stability, and usually code to rRNA molecules The region denoted by the circle corresponds to an area near the origin with lower curvature, stacking energy and position preference right before another area with greater flexibility.

10. Zoom of Genome Atlas, where only some of the previous features are shown. The first line shows position preference, the green stripes, show the low position preference, that are most likely to correspond to highly expressed genes. note the dark green stripes. It can be seen that sometimes the zones of the genome corresponding to the low position preference don�t match with RNA (line B, green or blue stripes) as it would be expected. That can may be caused, for instance because of low stabilty of RNA that is rapidly decomposed and don�t appears in the results. Also, we can see that green zones correspond to lower AT, higher stability, and purple zones to higher AT, lower stability and greater flexibilityZoom of Genome Atlas, where only some of the previous features are shown. The first line shows position preference, the green stripes, show the low position preference, that are most likely to correspond to highly expressed genes. note the dark green stripes. It can be seen that sometimes the zones of the genome corresponding to the low position preference don�t match with RNA (line B, green or blue stripes) as it would be expected. That can may be caused, for instance because of low stabilty of RNA that is rapidly decomposed and don�t appears in the results. Also, we can see that green zones correspond to lower AT, higher stability, and purple zones to higher AT, lower stability and greater flexibility

11. These are genome maps for the two plasmids Global repeat areas show possible sites for homologous recombination or insertion elementsThese are genome maps for the two plasmids Global repeat areas show possible sites for homologous recombination or insertion elements

12. In the article we based this presentation on, the authors have compared saprophyticus genome with 2 other staphylococci, aureus and epidermidis, in order to evaluate the pathogenesis of uncomplicated urinary tract infection in our bacteria. In the article we based this presentation on, the authors have compared saprophyticus genome with 2 other staphylococci, aureus and epidermidis, in order to evaluate the pathogenesis of uncomplicated urinary tract infection in our bacteria.

13. We can see that all the three genome are very similar in terms of AT content and size We can see that all the three genome are very similar in terms of AT content and size

14. Very similar origins of replication in both S. Aureus and S. Saprophyticus Very similar origins of replication in both S. Aureus and S. Saprophyticus

15. - The comparative genomic analyses between S. Epidermidis, S. Aureus and S. Saprophyticus, as well as the comparison with other uropathogenic bacteria and uropathogenic factors allowed the uncovering the mechanisms behind urinary tract infection by the saprophyticus bacteria.- The comparative genomic analyses between S. Epidermidis, S. Aureus and S. Saprophyticus, as well as the comparison with other uropathogenic bacteria and uropathogenic factors allowed the uncovering the mechanisms behind urinary tract infection by the saprophyticus bacteria.

16. We are familiar with the S. Aureus virulennce factors and mechanisms that allow this bacteria to survive inside the human body and cause damage to the host tissues when compared to the S. Epidermidis bacteria from which a lot less is know We are familiar with the S. Aureus virulennce factors and mechanisms that allow this bacteria to survive inside the human body and cause damage to the host tissues when compared to the S. Epidermidis bacteria from which a lot less is know

18. Genome size: 2,516,575bp GC content: 33.2%. Different functions are assigned to different colors in the diagram. Remember to refer the original and terminus similar to the other two bacteria.Genome size: 2,516,575bp GC content: 33.2%. Different functions are assigned to different colors in the diagram. Remember to refer the original and terminus similar to the other two bacteria.

19. S. saprophyticus ATCC 15305 genome characteristics - results

20. Everything is pretty much similarEverything is pretty much similar

21. This subtitle must be wrong!!!!!!!!!!!!!!!!!!!!This subtitle must be wrong!!!!!!!!!!!!!!!!!!!!

22. Check genes in page 13274. Complete slide SCC15305RM contains no virulence factors or antibiotic resistance. SCC15305cap S. aureus-type I cap enzymes, 3 more putative glycosyl transferases and a putative polysaccharide polymerase. These cassettes do not contain antibiotic resistance genes like similar cassettes found in S. aureusCheck genes in page 13274. Complete slide SCC15305RM contains no virulence factors or antibiotic resistance. SCC15305cap S. aureus-type I cap enzymes, 3 more putative glycosyl transferases and a putative polysaccharide polymerase. These cassettes do not contain antibiotic resistance genes like similar cassettes found in S. aureus

23. Orthologous vs Paralogous Homologous sequences are orthologous if they were separated by a speciation event: if a gene exists in a species, and that species diverges into two species, then the copies of this gene in the resulting species are orthologous; Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated, then the two copies are paralogous;

24. Refer that this is the number of predicted ORFs. 582 (23.8%) of the ORFs are specific to saprophyticus (most of them classified as physiological functions, like energy production and transcription ) Low number of virulence factors and mobile elements for saprophyticus. All virulence factors that aureus has are absent in saprophyticus.Refer that this is the number of predicted ORFs. 582 (23.8%) of the ORFs are specific to saprophyticus (most of them classified as physiological functions, like energy production and transcription ) Low number of virulence factors and mobile elements for saprophyticus. All virulence factors that aureus has are absent in saprophyticus.

25. Sacrophyticus has developed extra paralogs of transport systems related to function in a urine environment, and the presence of a cell wall-anchored protein used for uropathogenicity. Also, the bacteria shows high urease activity as determined by the quantitative measurement of ammonia when growing on Christensen urea agar. NRAMP is required in order to obtain iron, this system is unique to saprophyticus. Sacrophyticus has developed extra paralogs of transport systems related to function in a urine environment, and the presence of a cell wall-anchored protein used for uropathogenicity. Also, the bacteria shows high urease activity as determined by the quantitative measurement of ammonia when growing on Christensen urea agar. NRAMP is required in order to obtain iron, this system is unique to saprophyticus.

26. Well 1 - aureus (negative) Well 2 - aureus with control vector pRIT5H Well 3 - aureus RN4220 with SSP0135 adhesin (positive) Well 4 - Sacrophyticus (positive control) Well 5 - Sacrophyticus with deletion of the cap cassette (positive) Well 6 - Sacrophyticus with deletion of the UafA adhesin gene (negative) UafA encodes for a specific adhesin required to adhere to the eukaryotic cell in the urinary tract. SSP0135 is the largest of all of the predicted ORFs on the genome. Well 1 - aureus (negative) Well 2 - aureus with control vector pRIT5H Well 3 - aureus RN4220 with SSP0135 adhesin (positive) Well 4 - Sacrophyticus (positive control) Well 5 - Sacrophyticus with deletion of the cap cassette (positive) Well 6 - Sacrophyticus with deletion of the UafA adhesin gene (negative) UafA encodes for a specific adhesin required to adhere to the eukaryotic cell in the urinary tract. SSP0135 is the largest of all of the predicted ORFs on the genome.

27. Summary of the results Paralog expansion of transport systems is related to highly variable ion contents in the urinary environment Capable of adhering because of the presence of the adhesin UafA Survival capabilities explained by the three last factors Criticize the article! Paralog expansion of transport systems is related to highly variable ion contents in the urinary environment Capable of adhering because of the presence of the adhesin UafA Survival capabilities explained by the three last factors Criticize the article!

28. S. Saprophyticus ATCC 15305 The absence of virulence factors as in S. Aureus explains why saprophyticus causes uncomplicated UTI and not severe bacterial infectious disease. The systems is due to the paralogous expanding of the genome - this is needed in order to balance the osmotic pressure created by the variable osmotic environment in urine. The excessive urease production which is uncommon to the other staph strains is needed in order to obtain ammonium from the urea. This is due to the fact that the adhesion is used to anchor on to the urinary tract and prevent it from getting washed out by the micturition. Metabolic enzymes make it possible for the bacteria to have a prompt and individual response in a metabolite-rich urine. Iron is essential for bacterial survival, NRAMP is the most efficient system to have for the uptake of divalent cations because it acts in a pH-dependent manner under acidic pH conditions, like that of a urine environmment. The plasmids have a putative water channel aquaporin Z gene, which allows the bacteria to express water channels, multicopying the plasmids offers an advantage in water transport, this complements paralog expanding.The absence of virulence factors as in S. Aureus explains why saprophyticus causes uncomplicated UTI and not severe bacterial infectious disease. The systems is due to the paralogous expanding of the genome - this is needed in order to balance the osmotic pressure created by the variable osmotic environment in urine. The excessive urease production which is uncommon to the other staph strains is needed in order to obtain ammonium from the urea. This is due to the fact that the adhesion is used to anchor on to the urinary tract and prevent it from getting washed out by the micturition. Metabolic enzymes make it possible for the bacteria to have a prompt and individual response in a metabolite-rich urine. Iron is essential for bacterial survival, NRAMP is the most efficient system to have for the uptake of divalent cations because it acts in a pH-dependent manner under acidic pH conditions, like that of a urine environmment. The plasmids have a putative water channel aquaporin Z gene, which allows the bacteria to express water channels, multicopying the plasmids offers an advantage in water transport, this complements paralog expanding.