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Paper aims to investigate if antagonism of S. aueus by P. aeruginosa could induce SasG-dependent aggregation, promoting coexistence in chronic wounds

Intro - chronic wounds

• Previous studies have suggested that P. aeruginosa coexists with and then kills S. aureus in late-stage co-culture

• paper proposes late stage coculture is possible due to an antagonistic interaction taking place in early stage

• approx 20 million individuals worldwide have chronic wounds

• infection frequently results in delayed healing of chronic wounds and increase in patient morbidity and mortality.

• Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria co-isolated from chronic wounds

Intro - SASG

- giant, cell-wall anchored surface protein G (SasG) - involved in S. aureus aggregation and biofilm formation - orthologous to the S. epidermidis accumulation-associated protein (Aap) -has A and B domain - Aggregation occurs following the cleavage of A domain from B

• Paper proposes processing by P. aeruginosa proteases

• encourages co- infection of P. aeruginosa and

S. aureus

Figure 1 ••

• aim: investigate whether P. aeruginosa could induce SasG-dependent aggregation

• B) suspended strains of MRSA in varying concentrations of PAO1 cell free supernatant
• measured absorbance at 600nm and calculated % aggregation
• D) extracted MRSA cell wall proteins after treatment with PAO1 supernatant
• used SDS- PAGE and Coomassie staining to assess SasG cleavage

"Fig. 1. S. aureus aggregation is SasG-dependent and induced by P. aeruginosa."

Figure 1

"(A) Graphical representation of the structure of S. aureus SasG."

Figure 1

B)

• Can see aggregation of ΔmgrA is induced by PAO1 supernatant
• Aggregation is inhibited in double mutant
• Intervals of percentages tested seems a bit random
• text is thick and difficult to read

C)

• Clear image
• can see aggregates forming at the bottom of the tube
• could have fit in "10%" next to "PAO1" to make it clearer

"B) Aggregation of S. aureus MW2 ΔmgrA and ΔmgrAΔsasG mutants following incubation for one hour with P. aeruginosa cell-free supernatant diluted in PBS in increasing concentrations from 0-100%. (C) Representative image showing aggregation of the ΔmgrA and ΔmgrAΔsasG mutants following incubation with 10% P. aeruginosa supernatant for one hour at room temperature."

Figure 1

D)

• Can see processing of sasG
• what is in the 0 lane ? -> buffer only ?
• units for the ladder ?
• Used purified protein as controls seen in the supplementary -> figure seems incomplete without this

• Could have compared these to WT S. aureus
• Could have included phenotypic resque of phenotype by expressing sasG in trans

"Coomassie stained SDS-PAGE gel showing SasG processing by increasing concentrations of P. aeruginosa supernatant. Cell wall proteins were extracted from the same samples as described above prior to treatment (input) and following aggregation"

+ info

Figure 2 ••

• aim: to determine which quarum sesnisng system of PAO1 was responsible for the secreation of the protein which cleaves sasG

• A, B) Generated quarum sensing mutants and incubated their supernatants with MRSA ΔmrgA - ΔlasR, ΔrhtR, Δ pqsA

• C) Diagram representing LasR quarum sensing system and cross regulation with RhiR

• D, E , F, G) single, double and triple mutants of proteins under control of LasR transcriptional activator

"Fig. 2. P. aeruginosa las regulated 998 proteases cleave SasG and induce S. aureus aggregation."

Figure 2

• (A) ΔlasR had depleted aggregation
• they do double knockout of only one combination -> do not explain why
• (D) LasB and AprA can act together to enhance proteolytic activity -> overprocess SasG

• text too thick again

• keep WT and PBS controls on the same side in A as D and F for continuity

• would want to see if there is a significant difference from WT POA1 but only compare to PBS

Figure 2

• C) Diagram shows LasR quorum sensing system but figure legend does not reflect this

• Do not tell the reader what AHL stands for

+ info

+ info

"(C) MRSA aggregation induced by a triple protease mutant ΔlasAΔlasBΔaprA. Each protease is sufficient to cleave SasG, but exhibits varying levels of activity."

Figure 3 (A) ••

• A) Used qPCR to asses the transcriptional expression of protease genes lasA, lasB, and aprA relative to gene rpoD in wild-type PAO1 and isogenic mutants of Δ rhlR and Δ lasR
• are showing significance to WT this time
• illegible

"Fig. 1. Figure 3. Protease genes lasA, lasB, and aprA are differentially expressed in P. aeruginosa PAO1 in a quorum sensing-dependent manner."

Figure 3 (B) ••

• B) previous work shows aprA is significantly upregulated in vivo and clinical wound specamins
• is aprA expression alone enough to cause aggregation ?

• expressed aprA in E.coli BL21 under an arabinose inducible promotor.
• no MRSA aggregation induced by wild type BL21
• 'pBAD18- aprA induced' caused aggregation in "similar levels" to wild type PAO1
• heterologous expression of AprA is enough to cause Sas-G dependent aggregation

"Fig. 1. Figure 3. Protease genes lasA, lasB, and aprA are differentially expressed in P. aeruginosa PAO1 in a quorum sensing-dependent manner."

Figure 4 ••

• aim: investigate whether S. aureus aggregates formed in response to PAO1 had an effect on their antimicrobial susceptibility and bacterial persistence

• Isolates were exposed to Ciprofloxacin (4A) and Vancomycin ( 4B) concentrations ranging from sublethal to 2-4 times the MIC.

• PAO1 supernatant facilitated survival of ΔmgrA bacteria
• ΔsasG mutant strain exhibited significantly lower colony forming units than ΔmgrA
• Higher CFUs in cells treated with PAO1 than in PBS (4B)

"Figure 4. SasG-dependent S. aureus aggregates exhibit increased tolerance to antibiotics Ciprofloxacin and Vancomycin."

Figure 4 ••

• Stripes were unnecessary
• illegible

"Figure 4. SasG-dependent S. aureus aggregates exhibit increased tolerance to antibiotics Ciprofloxacin and Vancomycin."

Figure 5

Aim: to evaluate the role of SasG in biofilm formation and early s. aureus/ POA1 interactions in wound like conditions in vitroA) Schematic of the Lubbock Biofilm model

• used wound like media (WLM) to replicate chronic wound environment in vitro

B) -MRSA ΔmgrA and the ΔsasG mutants were inoculated into WL as either mono- or co-infections with PAO1 and incubated for 24 hours

• representative images of biofilms formed

C) CFU of MRSA ΔmgrA and the ΔsasG and PAO1 after antibiotic treatment D) Inoculated fluorescent strains of MRSA and PAO1 with WLM, harvested biofilms and observed using confocal laser scanning microscopy E, F, G, H - thickness, biomass thickness, biovolume

"Figure 5. SasG-dependent aggregates promote 1036 biofilm formation and contribute to S. aureus survival when co-infected with P. aeruginosa in vitro."

Figure 5 ••

A) - figure itself is very clear apart from yellow droplet B) clear image, but is glazed over C) - no difference in survival amongst monomicrobial biofilms

• Polymicrobial biofilms (ΔmgrA and PAO1) had little difference in cell viability between the two pathogens, - both exhibited slight increases in CFUs compared to the monomicrobial biofilms

• Polymicrobial biofilms with the ΔsasG mutant had a significant decrease in MRSA cell viability and an increase in PAO1

-> lack of comparability -> could have followed similar layout to figure 3A

"(A)Schematic of the Lubbock Biofilm model.."

Figure 3 (A)

Figure 5 ••

• (D) - mgrA mutant – dense robust aggregates throughout the biommass

• sasG mutant- had difficulty identifying MRSA in the biofilm and those identifiable were in distinct niches at the periphery of the biofilm separated from PA

(E, F, G, H)

• measuring average thickness and thickness of biomass
• fixed the weird graphs
• how did they measure the thickness ? -> lacking in methods

• disrupting biofilm by taking it from a pipette tip ?
• questionable accuracy of thickness

VERSUS

Example

I. Ahmad et al., “Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii,” npj Biofilms and Microbiomes, vol. 9, no. 1, Dec. 2023, doi: https://doi.org/10.1038/s41522-023-00465-6.

Figure 6

Aim : to investigate the impact of SasG dependent biofilm formation on S. aureus survival during co-infection with P. aeruginosa in vivo A) Schematic of in vivo polymicrobial chronic wound model B) Representative images showing chronic wound progression over the 9 day time course. C) Quantification of wound healing over 9 days represented as the percent difference of the initial wound size with statistical significance representing comparisons between co-infections. D) MRSA and (E) PAO1 CFUs recovered from excised wound tissue at the day 9 endpoint. Results represent an average of three independent experiments performed

"Figure 6. SasG increases S. aureus survival and contributes t 1053 o worse clinical outcomes in an in vivo model of polymicrobial chronic wound infections."

Figure 6

A) nicely layed out, clear and easy to understand B) images are very clear but do not have scale bars so the size of the wounds is not comparable from the figure alone

• measured images in imageJ

C) starts from day 1 but A states that imaging occurs from day 2 onwards D, E) - squiched text "co- infection" "mono-infection"

• inconsistent "ns"

Conclusions

• - P. aeruginosa induces SasG-dependent aggregation in vitro

• LasR quorum sensing system responsible for the regulation of proteases: LasA, LasB, AprA, which process SasG

• increase in aggregation when SasG is processed

• processing of SasG by POA1 causes thicker biofilm formation in wound like conditions in vitro

• SasG dependent biofilm formation causes increase in S. aureus survival during co-infection with P. aeruginosa in vivo

THANKS !!

Refrences

[1]D. Leaper, O. Assadian, and C. E. Edmiston, “Approach to Chronic Wound Infections,” British Journal of Dermatology, vol. 173, no. 2, pp. 351–358, Mar. 2015, doi: https://doi.org/10.1111/bjd.13677. [2]A. R. Siddiqui and J. M. Bernstein, “Chronic wound infection: Facts and controversies,” Clinics in Dermatology, vol. 28, no. 5, pp. 519–526, Sep. 2010, doi: https://doi.org/10.1016/j.clindermatol.2010.03.009. [3]R. Serra et al., “Chronic wound infections: the role ofPseudomonas aeruginosaandStaphylococcus aureus,” Expert Review of Anti-infective Therapy, vol. 13, no. 5, pp. 605–613, Mar. 2015, doi: https://doi.org/10.1586/14787210.2015.1023291. [4]L. M. Filkins et al., “Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model,” Journal of Bacteriology, vol. 197, no. 14, pp. 2252–2264, Apr. 2015, doi: https://doi.org/10.1128/jb.00059-15. [5]M. Di Giulio et al., “Graphene Oxide affects Staphylococcus aureus and Pseudomonas aeruginosa dual species biofilm in Lubbock Chronic Wound Biofilm model,” Scientific Reports, vol. 10, no. 1, Oct. 2020, doi: https://doi.org/10.1038/s41598-020-75086-6. [6]I. Ahmad et al., “Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii,” npj Biofilms and Microbiomes, vol. 9, no. 1, Dec. 2023, doi: https://doi.org/10.1038/s41522-023-00465-6.

• First and only time figure 2C is referenced
• also does not relate to figure legend

• supplementary figure 1B
• has controls
• " quantified" but numbers in the boxes are too small to read

• pacman image slightly covering LasB text