Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa.

IMPORTANCE: Polymicrobial infections are common. In chronic infections, the different pathogens may repeatedly interact, which could spur evolutionary dynamics with pathogens adapting to one another. Here, we explore the potential of Staphylococcus aureus to adapt to its competitor Pseudomonas aeruginosa. These two pathogens frequently co-occur, and P. aeruginosa is seen as the dominant species being able to displace S. aureus. We studied three different S. aureus strains and found that all became quickly resistant to inhibitory compounds secreted by P. aeruginosa. Our experimental evolution revealed strains-specific adaptations with three main factors contributing to resistance evolution: (i) overproduction of staphyloxanthin, a molecule protecting from oxidative stress; (ii) the formation of small colony variants also protecting from oxidative stress; and (iii) alterations of membrane transporters possibly reducing toxin uptake. Our results show that species interactions can change over time potentially favoring species co-existence, which in turn could affect disease progression and treatment options.

Single-Cell Imaging Reveals That Staphylococcus aureus Is Highly Competitive Against Pseudomonas aeruginosa on Surfaces.

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and their interactions can complicate disease progression and treatment options. While interactions between P. aeruginosa and S. aureus have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important because both species frequently colonize surfaces to form aggregates and biofilms in infections. Here, we performed single-cell time-lapse fluorescence microscopy, combined with automated image analysis, to describe interactions between P. aeruginosa PAO1 with three different S. aureus strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While P. aeruginosa is usually considered the dominant species, we found that the competitive balance tips in favor of S. aureus on surfaces. We observed that all S. aureus strains accelerated the onset of microcolony growth in competition with P. aeruginosa and significantly compromised P. aeruginosa growth prior to physical contact. Upon direct contact, JE2 was the most competitive S. aureus strain, simply usurping P. aeruginosa microcolonies, while 6850 was the weakest competitor itself suppressed by P. aeruginosa. Moreover, P. aeruginosa reacted to the assault of S. aureus by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.

Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery.

The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non-virulent and a non-replicating, virulent/transmissive phase. Here, we show on a single-cell level that at late stages of infection, individual motile (PflaA -GFP-positive) and virulent (PralF - and PsidC -GFP-positive) L. pneumophila emerge in the cluster of non-growing bacteria within an LCV. Comparative proteomics of PflaA -GFP-positive and PflaA -GFP-negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA -GFP-positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi-phasic life cycle of L. pneumophila.

Strain Background, Species Frequency, and Environmental Conditions Are Important in Determining Pseudomonas aeruginosa and Staphylococcus aureus Population Dynamics and Species Coexistence.

Bacterial communities in the environment and in infections are typically diverse, yet we know little about the factors that determine interspecies interactions. Here, we apply concepts from ecological theory to understand how biotic and abiotic factors affect interaction patterns between the two opportunistic human pathogens Pseudomonas aeruginosa and Staphylococcus aureus, which often cooccur in polymicrobial infections. Specifically, we conducted a series of short- and long-term competition experiments between P. aeruginosa PAO1 (as our reference strain) and three different S. aureus strains (Cowan I, 6850, and JE2) at three starting frequencies and under three environmental (culturing) conditions. We found that the competitive ability of P. aeruginosa strongly depended on the strain background of S. aureus, whereby P. aeruginosa dominated against Cowan I and 6850 but not against JE2. In the latter case, both species could end up as winners depending on conditions. Specifically, we observed strong frequency-dependent fitness patterns, including positive frequency dependence, where P. aeruginosa could dominate JE2 only when common (not when rare). Finally, changes in environmental (culturing) conditions fundamentally altered the competitive balance between the two species in a way that P. aeruginosa dominance increased when moving from shaken to static environments. Altogether, our results highlight that ecological details can have profound effects on the competitive dynamics between coinfecting pathogens and determine whether two species can coexist or invade each others' populations from a state of rare frequency. Moreover, our findings might parallel certain dynamics observed in chronic polymicrobial infections.IMPORTANCE Bacterial infections are frequently caused by more than one species, and such polymicrobial infections are often considered more virulent and more difficult to treat than the respective monospecies infections. Pseudomonas aeruginosa and Staphylococcus aureus are among the most important pathogens in polymicrobial infections, and their cooccurrence is linked to worse disease outcome. There is great interest in understanding how these two species interact and what the consequences for the host are. While previous studies have mainly looked at molecular mechanisms implicated in interactions between P. aeruginosa and S. aureus, here we show that ecological factors, such as strain background, species frequency, and environmental conditions, are important elements determining population dynamics and species coexistence patterns. We propose that the uncovered principles also play major roles in infections and, therefore, proclaim that an integrative approach combining molecular and ecological aspects is required to fully understand polymicrobial infections.

Mms19 is a mitotic gene that permits Cdk7 to be fully active as a Cdk-activating kinase.

Mms19 encodes a cytosolic iron-sulphur assembly component. We found that Drosophila Mms19 is also essential for mitotic divisions and for the proliferation of diploid cells. Reduced Mms19 activity causes severe mitotic defects in spindle dynamics and chromosome segregation, and loss of zygotic Mms19 prevents the formation of imaginal discs. The lack of mitotic tissue in Mms19P/P larvae can be rescued by overexpression of the Cdk-activating kinase (CAK) complex, an activator of mitotic Cdk1, suggesting that Mms19 functions in mitosis to allow CAK (Cdk7/Cyclin H/Mat1) to become fully active as a Cdk1-activating kinase. When bound to Xpd and TFIIH, the CAK subunit Cdk7 phosphorylates transcriptional targets and not cell cycle Cdks. In contrast, free CAK phosphorylates and activates Cdk1. Physical and genetic interaction studies between Mms19 and Xpd suggest that their interaction prevents Xpd from binding to the CAK complex. Xpd bound to Mms19 therefore frees CAK complexes, allowing them to phosphorylate Cdk1 and facilitating progression to metaphase. The structural basis for the competitive interaction with Xpd seems to be the binding of Mms19, core TFIIH and CAK to neighbouring or overlapping regions of Xpd.