Stenotrophomonas maltophilia affects the gene expression profiles of the major pathogens Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro multispecies biofilm model

Abstract

In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of Stenotrophomonas maltophilia on its commensals Pseudomonas aeruginosa and Staphylococcus aureus in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.

Keywords: mixed species biofilms; pathogens; transcriptome analysis.

Fig 1

Laser scanning microscope images of fluorescent-labeled species used in this study. The different species S. maltophilia and P. aeruginosa were labeled with the mini Tn7T transposon. The strain S. aureus SH1000 was labeled with the plasmid PCM29 coding the fluorescent proteins under the SarA promotor. C. albicans SC5314 was labeled by integrating the fluorescent protein into the chromosome. Panel A shows images of S. maltophilia labeled with different fluorescent proteins. Panel B shows P. aeruginosa PAO1 labeled with different fluorescent proteins. Panel C shows images of S. aureus SH1000 labeled with different fluorescent proteins. Panel D shows C. albicans SC5314 labeled with different fluorescent proteins. In panel E, the principle of the Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins is demonstrated. Panel F shows a part of the plasmid PCM29 encoding the fluorescent proteins under the SarA promotor. This vector was used for tagging the strain S. aureus SH1000.

Fig 2

Dual-species biofilms confocal analyses and layer formation. Dual-species biofilms were grown under static conditions at 37°C in 10% LB and analyzed with confocal microscopy. Images were taken after 24 h, 48 h, and 72 h. Dual-species biofilm of S. maltophilia K279a tdTomato (red) + S. aureus SH1000 eCyan (cyan) (A), S. maltophilia K279a tdTomato (red) + P. aeruginosa PA01 GFP (green) (B), S. maltophilia K279a tdTomato (red) + C. albicans SC5314 sfGFP (green) (C), S. aureus SH1000 mCherry (red) + C. albicans SC5314 sfGFP (green) (D), P. aeruginosa PA01 mCherry (red) + C. albicans SC5314 sfGFP (green) (E), and P. aeruginosa PA01 GFP (green) + S. aureus SH1000 mCherry (red) (F). In (C1 + 2), the attachment of S. maltophilia K279a tdTomato (red) on the hyphae of C. albicans SC5314 sfGFP (green) is shown after 48 h and 72 h. (G) Dual-species biofilm of S. maltophilia K279a sfGFP (green) and P. aeruginosa mCherry (red) grown under flow conditions for 72 h at 28°C. Image analysis shows a more pronounced layer formation. .

Fig 3

Confocal microscopy images of triple-species biofilms were grown under static conditions at 37°C in 10% LB. Images were taken after 24 h, 48 h, and 72 h. (A) Triple-species biofilm of S. maltophilia K279a tdTomato (red) + S. aureus SH1000 AmCyan (cyan) + C. albicans SC5314 sfGFP (green). (B) Triple-species biofilm of S. maltophilia K279a tdTomato (red) + P. aeruginosa PAO1 eYFP (yellow) + C. albicans SC5314 sfGFP (green). (C) Triple-species biofilm of S. maltophilia K279a tdTomato (red) + S. aureus SH1000 AmCyan (cyan) + P. aeruginosa PAO1 GFP (green).

Fig 4

Mean total volume of single-, dual-, and triple-species biofilms indicating species and time-dependent growth behavior. Biofilms were cultivated in LB 10% with a start OD of 0.05 in Ibidi chamber slides under static conditions. After 24 h, 48 h, and 72 h at 37°C, the mean pixel volume of three samples per species was calculated as described in Materials and Methods. *P-value <0.05; **P-value <0.001.

Fig 5

Volcano plots representing the differential gene expression results of RNAseq data. Upregulated genes (log2 fold change ≥2) are represented by red dots, whereas downregulated genes (log2-fold change ≤−2) are represented by blue dots. (A) K279a + SH1000: regulated genes of K279a belong to the lactate metabolism [SMLT_RS13840 (A1), SMLT_RS13835 (A2)] and the glyoxylate cycle [SMLT_RS01085 (A3), SMLT_RS01090 (A4)]. (B) K279a + SH1000 + SC5314: regulated genes of K279a belong to the lactate metabolism [SMLT_RS13840 (B1), SMLT_RS13835 (B2), SMLT_RS13830 (B3)] and the glyoxylate cycle [SMLT_RS01090 (B4), SMLT_RS01085 (B5)]. (C) K279a + PAO1: regulated genes of K279a belong to the shape [SMLT_RS19285 (C1)], propionate degradation [SMLT_RS17185 (C2)], secretion systems [SMLT_RS13065 (C3), SMLT_RS06235 (C4)], respiration [SMLT_RS20930 (C5)], the glyoxylate cycle [SMLT_RS01090 (C6), SMLT_RS01085 (C7)], and the tryptophane biosynthesis [SMLT_RS16260 (C8)]. (D) K279a + PAO1 + SC5314: regulated genes of K279a are a DcaP family trimeric outer membrane transporter [SMLT_RS21910 (D1)], a propionate–CoA ligase [SMLT_RS04515 (D2)]. (E) K279a + SC5314: regulated genes of K279a are a DcaP family trimeric outer membrane transporter [SMLT_RS21910 (E1)] and a propionate–CoA ligase [SMLT_RS04515 (E2)]. (F) SH1000 + K279a: regulated genes of SH1000 are a serine/threonine exchange transporter [SAOUHSC_01450 (F1)], the transcriptional regulator sarV [SAOUHSC_02532 (F2)], a toxin called YoeB [SAOUHSC_02691 (F3)] and the tryptophane biosynthesis [SAOUHSC_01369 (F4), SAOUHSC_01368 (F5), SAOUHSC_01371 (F6), SAOUHSC_01372 (F7)]. (G) SH1000 + K279a + SC5314: regulated genes of SH1000 are a serine/threonine exchange transporter [SAOUHSC_01450 (G1)], the transcriptional regulator sarV [SAOUHSC_02532 (G2)], and the tryptophane biosynthesis [SAOUHSC_01371 (G3), SAOUHSC_01372 (G4), SAOUHSC_01368 (G5), SAOUHSC_01369 (G6)]. (H) PAO1 + K279a: regulated genes of PAO1 are a thioestherase [PA1019a (H1)], type 2 [PA2676 (H2)], type 3 [PA1698 (H3), PA1724 (H4)], and type 6 [PA5090 (H5)] secretion systems, alginate biosynthesis [PA3549 (H6), PA3546 (H7)], exotoxin A [PA1148 (H8)], virulence factors [PA3724 (H9), PA1871 (H10)], and quorum-sensing-related genes [PA1432 (H11), PA2227 (H12), PA3476 (H13)]. (I) PAO1 + K279a + SC5314: regulated genes of PAO1 are a thioestherase [PA1019a (I1)], type 2 [PA0684 (I2)], and type 3 [PA1718 (I3)] secretion systems, type III export protein PscG (I4), virulence factors [PA3724 (I5), PA1871 (I6)], transcriptional regulator RhlR (I7) quorum-sensing-related genes [PA2227 (I8), PA1432 (I9)], and genes involved in aerobic respiration [PA0105 (I10), PA0108 (I11), PA0106 (I12)].

Fig 6

Promotor fusion construct pBBR1MCS::P4401::mCerulean::P1360::GFP reveals different respiration systems in S. maltophilia K279a in single versus co-culture. (A) Genetic map of the promotor fusion construct pBBR1MCS::P4401::mCerulean::P1360::GFP. When growing alone, only Cyt 1360 is strongly expressed in K279a (BI, BII), whereas in co-biofilm with PAO1, the Cyt 4401 is additionally also highly expressed (BIII, BIV). (C) Dual-species biofilm of K279a × pBBR1MCS::P4401::mCerulean and PAO1 mCherry. Cyt 4401 (blue) is highly expressed, and the mCerulean signal correlates with the layer of K279a in the middle of the biofilm.

Fig 7

Possible interactions between the different species used in this study. RNAseq analysis of biofilms, which were cultivated in 10% LB in Ibidi slide chambers at 37°C for 72 h, revealed a differentially gene expression of each species in dual- and triple-species biofilms as compared to single-species biofilms.