From the wound to the bench: exoproteome interplay between wound-colonizing Staphylococcus aureus strains and co-existing bacteria

Abstract

Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors.

Keywords: Bacillus thuringiensis; Klebsiella oxytoca; Staphylococcus aureus; chronic wound; co-culture; exoproteome.

Figure 1.

Growth characteristics of the isolated bacteria on agar plates. (A) Microbiome topography of culturable bacteria from the wound dressing of a patient with Junctional EB. The used dressing from a chronic wound was replica plated onto CLED agar for isolation of the bacteria present. (B) Zone inhibition experiments. Unimpaired staphylococcal growth upon spotting of B. thuringiensis or K. oxytoca: (i) t111+Bt, (ii) t13595+Bt, (iii) t111+Ko, and (iv) t13595+Ko. Unimpaired growth of K. oxytoca upon spotting of B. thruingiensis (v). S. aureus growth inhibition halo caused by spotting of B. subtilis 168 onto a lawn of staphylococcal cells (vi; both S. aureus strains exhibited the same effect). (C) Dilution and subsequent plating: (i) S. aureus colonies growing on top of larger colonies of B. thuringiensis; (ii) K. oxytoca and S. aureus colonies growing in close proximity and occasionally touching each other. Typical S. aureus colonies are marked with arrows.

Figure 2.

Total numbers of extracellular proteins identified per culture. (A) Numbers of S. aureus t111 and t13595 exoproteins identified in monocultures or upon co-culturing with B. thuringiensis (+Bt) or K. oxytoca (+Ko). (B) Numbers of B. thuringiensis and K. oxytoca exoproteins identified in monocultures or upon co-culturing with S. aureus t111 (+t111) or t13595 (+t13595) isolates. (C-F) Predicted subcellular localization of proteins per strain in mono- or co-cultures. (C) 11, S. aureus t111 exoproteins; 11B, S. aureus t111 exoproteins upon co-culture with B. thuringiensis; 11K, S. aureus t111 exoproteins upon co-culture with K. oxytoca. (D) 13, S. aureus t13595 exoproteins; 13B, S. aureus t13595 exoproteins upon co-cultures with B. thuringiensis; 13K, S. aureus t13595 exoproteins upon co-culture with K. oxytoca. (E) K, K. oxytoca exoproteins; K11, K. oxytoca exoproteins upon co-culture with S. aureus t111; K13, K. oxytoca exoproteins upon co-culture with S. aureus t13595. (F) B, B. thuringiensis exoproteins; B11, B. thuringiensis exoproteins upon co-culture with S. aureus t111; B13, B. thuringiensis exoproteins upon co-culture with S. aureus t13595.

Figure 3.

Relationships among cultures. The diagrams show the number of proteins identified in all cultures and those proteins shared in the different conditions. (A) Total number of S. aureus t111 proteins in monoculture (11) and in co-culture with B. thuringiensis (11B) or K. oxytoca (11K); (B) Total number of S. aureus t13595 proteins in monoculture (13) and in co-culture with B. thuringiensis (13B) or K. oxytoca (13K); (C) Total number of B. thuringiensis proteins in monoculture (B) and in co-culture with S. aureus t111 (B11) or S. aureus t13595 (B13); (D) Total number of K. oxytoca proteins in monoculture (K) and in co-culture with S. aureus t111 (K11) or S. aureus t13595 (K13); (E) Total number of S. aureus proteins in monoculture (11) or (13) and in co-culture (11+13).

Figure 4.

Overview of the predicted functions of the exoproteins found in all staphylococcal cultures. Diagram (A) depicts only S. aureus t111 proteins. The area on the top shows the proteins detected only in monoculture, while the lower left area shows a single protein detected when t111 was co-cultured with B. thuringiensis. Likewise, diagram (B) shows exclusively the proteins that belong to S. aureus t13595 in monoculture and co-culture with B. thuringiensis or K. oxytoca. Diagram (C) depicts the proteins of S. aureus t111 and t13595 grown in monoculture in the bottom areas, whilst the upper area shows the proteins identified upon co-culturing both S. aureus isolates. In the latter area, the circles are represented as pie charts. The charts show the percentage of proteins that belong to each isolate. For the unknown proteins (in grey), 50% belong to t111, 37% to t13595, and 12.5% to both of them; for the cell envelope proteins (blue) 67% belong to t111 and 33% to both isolates; for proteins involved in intermediary metabolism (green), 25% belong to t111 and 75% to t13595; and for information pathways proteins (yellow), 50% belong to t111 and 50% to both isolates.