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Many human microbiome members inhibit bacterial competitors by production of antimicrobial compounds whose expression needs to be tightly controlled to balance the costs and benefits of compound biosynthesis. The nasal commensal Staphylococcus lugdunensis outcompetes Staphylococcus aureus using the antimicrobial lugdunin. The lugdunin biosynthetic gene cluster (BGC) encodes two potential regulators whose roles have remained unknown. Deletion of the regulator genes lugR or lugJ led to increased lugdunin production and/or immunity. While LugR was found to repress the transcription of the biosynthetic lugRABCTDZ operon, LugJ repressed the lugIEFGH export and immunity genes. Both regulators bound to different inverted repeats in the controlled promoter regions. Notably, both repressors were released from cognate promoters to allow transcription upon addition of exogenous lugdunin. Even minor structural changes disabled lugdunin derivatives to induce expression of its BGC, which is consistent with inferior binding to the predicted LugR and LugJ binding pockets. Thus, lugdunin controls its own biosynthesis through a feed-forward mechanism probably to avoid futile production.IMPORTANCEBiosynthetic gene clusters (BGCs) are usually tightly controlled to avoid production of costly goods at inappropriate time points or unfavorable conditions. However, in most cases, the regulatory signals of these clusters have remained unknown. Frequently, quorum sensing or two-component regulatory systems are involved in BGC expression control. This study elucidates the sophisticated regulation of lugdunin biosynthesis and secretion via two independent regulators, LugR and LugJ. Although belonging to different families of repressors, both directly interact with the antimicrobial lugdunin and thereby enhance biosynthesis and secretion in a feed forward-like mechanism.

The human pathogenic fungus Aspergillus fumigatus establishes dual biofilm interactions in the lungs with the pathogenic bacterium Pseudomonas aeruginosa. Screening of 21 A. fumigatus null mutants revealed seven mutants (two G protein-coupled receptors, three mitogen-activated protein kinase receptors, a Gα protein, and one histidine kinase receptor) with reduced biofilm formation, specifically in the presence of P. aeruginosa. Transcriptional profiling and metabolomics analysis of secondary metabolites produced by one of these mutants, ΔgpaB (gpaB encodes a Gα protein), showed GpaB controls the production of several important metabolites for the dual biofilm interaction, including pyripyropene A, a potent inhibitor of mammalian acyl-CoA cholesterol acyltransferase. Deletion of pyr2, encoding a non-reducing polyketide synthase essential for pyripyropene biosynthesis, showed reduced A. fumigatus Δpyr2-P. aeruginosa biofilm growth, altered macrophage responses, and attenuated mouse virulence in a chemotherapeutic murine model. We identified pyripyropene as a novel player in the ecology and pathogenic interactions of this important human fungal pathogen.IMPORTANCEAspergillus fumigatus and Pseudomonas aeruginosa are two important human pathogens. Both organisms establish biofilm interactions in patients affected with chronic lung pulmonary infections, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease. Colonization with A. fumigatus is associated with an increased risk of P. aeruginosa colonization in CF patients, and disease prognosis is poor when both pathogens are present. Here, we identified A. fumigatus genetic determinants important for the establishment of in vitro dual A. fumigatus-P. aeruginosa biofilm interactions. Among them, an A. fumigatus Gα protein GpaB is important for this interaction controlling the production of the secondary metabolite pyripyropene. We demonstrate that the lack of pyripyropene production decreases the dual biofilm interaction between the two species as well as the virulence of A. fumigatus in a chemotherapeutic murine model of aspergillosis. These results reveal a complete novel role for this secondary metabolite in the ecology and pathogenic interactions of this important human fungal pathogen.

Piscirickettsia salmonis is a globally distributed aquatic bacterium and a component of the normal salmon microbiome. It has significant biological and economic impact on Chilean salmon aquaculture due to the highly fatal disease, piscirickettsiosis. Unsuccessful attempts to prevent and treat this disease have resulted in heavy use of antimicrobials with adverse effects on the aquatic environment and piscine and human health. Evidence suggests P. salmonis could be a bacterium with relative pathogenic potential on farmed salmonids and other fishes that triggers piscirickettsiosis under particular conditions in the salmon and its environment. Application of a damage-response framework analysis could define the steps from asymptomatic P. salmonis infection to symptomatic disease, help tailor improved approaches to disease prevention and management, and, in turn, help avoid heavy use of antimicrobials which have global effects on animal health, human health, and environmental biodiversity (the One Health concept).

Powdery mildew is a global threat to crops and economically valuable plants. Salicylic acid (SA) signaling plays a significant role in plant resistance to biotrophic parasites; however, the mechanisms behind how powdery mildew fungi circumvent SA-mediated resistance remain unclear. Many phytopathogenic microbes deliver effectors into the host to sustain infection. In this study, we showed that the rubber tree powdery mildew fungus Erysiphe quercicola inhibits host SA biosynthesis by employing two effector proteins, EqCmu and EqPdt. These effector proteins can be delivered into plant cells to hydrolyze chorismate, the main precursor of SA, through their enzymatic activities. Notably, EqCmu and EqPdt can interact with each other, providing mutual protection against protein degradation mediated by the plant ubiquitin-proteasome system. This interaction enhances their activities in the hydrolysis of chorismate. Our study reveals a new pathogenic strategy by which two powdery mildew effector proteins cooperate to evade recognition by dampening the host immune system.

Importance: Powdery mildew fungi may develop diverse strategies to disturb salicylic acid (SA) signaling in plants, which plays an important role in activating immunity, and little is known about these strategies. Our results suggest that the Erysiphe quercicola effector protein EqCmu can be translocated into host cells and inhibit host SA levels during the infection stage; however, it is targeted by the plant ubiquitin-proteasome system (UPS) and ubiquitinated, which induces EqCmu degradation. To evade the UPS, EqCmu interacts with EqPdt, another E. quercicola effector protein, to prevent that ubiquitination. EqPdt also inhibits host SA biosynthesis through its prephenate dehydratase activity. Taken together, these two powdery mildew effector proteins cause a synergistic effect in disturbing host SA signaling. Our study also suggests that enhancing SA signaling is required for boosting immunity against powdery mildew fungus.

Genetically engineered bacteria represent a promising drug delivery tool for disease treatment. The development of new strategies for specific and independent protein regulation is necessary, especially for combination protein drug therapy. Using the well-studied Escherichia coli phage λ as a model system, we applied noncanonical amino acids (ncAAs) as novel inducers for protein regulation in a bacteria-based delivery system. Screening the permissive sites of the Cro protein revealed that incorporation of AlocK at the K8 site with the MbPylRS-349F/tRNA^Pyl system produced a functional Cro-K8AlocK variant. Using an engineered λ lysogen expressing the MbPylRS-349F/tRNA^Pyl pair, Cro-8X, and the reporter mNeonGreen, in vitro and in vivo experiments showed that AlocK led to bacterial lysis through prophage activation and the release of mNeonGreen. If mNeonGreen was integrated into the λ prophage genome, λ phages released due to AlocK induction delivered the reporter gene into the recipient E. coli strain, enabling mNeonGreen expression. Furthermore, insertion of pIF at the F14 site with the AfpIFRS/tRNA^Tyr pair produced a functional Cro-F14pIF variant. Importantly, AfpIFRS/tRNA^Tyr and MbPylRS-349F/tRNA^Pyl pairs were confirmed to be mutually orthogonal. In a mixture of two engineered λ lysogens expressing different aaRS/tRNAs, Cro-ncAAs, and reporter proteins, AlocK and pIF independently induced bacterial lysis and activated the expression of mNeonGreen and mCherry in the recipient E. coli strain. Collectively, the proposed bacteria-based delivery system provides two options for protein delivery and enables independent regulation of multiple proteins with ncAAs, offering a novel approach for in situ protein regulation and combination therapy.

Importance: The use of genetically engineered bacteria as drug delivery vectors has attracted more and more attention in recent years. A key issue with bacteria-based delivery systems is how to regulate multiple protein drugs. Based on genetic code expansion technology, we developed a new strategy of using ncAAs as small molecular inducers for in situ protein regulation and engineered λ phage lysogen into a bacteria-based delivery system that can function in two delivery modes. Furthermore, this strategy enables independent regulation of multiple proteins by different ncAAs, offering important implications for combination therapy. This approach requires minimal genetic engineering efforts, and similar strategies can be applied to engineer other prophage-bacteria systems or study phage biology. This work expands the therapeutic applications of ncAAs and lysogenic phages.

Mycobacterium abscessus is one of the leading causes of pulmonary infections caused by non-tuberculous mycobacteria. The ability of M. abscessus to establish a chronic infection in the lung relies on a series of adaptive mutations impacting, in part, global regulators and cell envelope biosynthetic enzymes. One of the genes under strong evolutionary pressure during host adaptation is ubiA, which participates in the elaboration of the arabinan domains of two major cell envelope polysaccharides: arabinogalactan (AG) and lipoarabinomannan (LAM). We here show that patient-derived UbiA mutations not only cause alterations in the AG, LAM, and mycolic acid contents of M. abscessus but also tend to render the bacterium more prone to forming biofilms while evading uptake by innate immune cells and enhancing their pro-inflammatory properties. The fact that the effects of UbiA mutations on the physiology and pathogenicity of M. abscessus were impacted by the rough or smooth morphotype of the strain suggests that the timing of their selection relative to morphotype switching may be key to their ability to promote chronic persistence in the host.IMPORTANCEMultidrug-resistant pulmonary infections caused by Mycobacterium abscessus and subspecies are increasing in the U.S.A. and globally. Little is known of the mechanisms of pathogenicity of these microorganisms. We have identified single-nucleotide polymorphisms (SNPs) in a gene involved in the biosynthesis of two major cell envelope polysaccharides, arabinogalactan and lipoarabinomannan, in lung-adapted isolates from 13 patients. Introduction of these individual SNPs in a reference M. abscessus strain allowed us to study their impact on the physiology of the bacterium and its interactions with immune cells. The significance of our work is in identifying some of the mechanisms used by M. abscessus to colonize and persist in the human lung, which will facilitate the early detection of potentially more virulent clinical isolates and lead to new therapeutic strategies. Our findings may further have broader biomedical impacts, as the ubiA gene is conserved in other tuberculous and non-tuberculous mycobacterial pathogens.

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturb this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitatively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored the contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a ΔexsD mutant "constitutively on" for T3SS expression was traversal defective. The dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except for the effector genes (ΔpscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented ΔpscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to the development of therapeutics targeting the T3SS system.IMPORTANCEWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of the mechanisms involved is derived largely from cell culture experimentation. The latter is due to the practical limitations of in vivo/in situ models and the challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by (i) leveraging the transparent properties and superficial location of the cornea, (ii) using our established method for enabling bacterial traversal susceptibility, and (iii) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Respiratory syncytial virus (RSV) was discovered in 1956 by the laboratory of Robert Chanock after its isolation from children with upper respiratory infections. Here, we review the events leading to its discovery including its prior isolation as chimpanzee coryza virus and its subsequent association with human disease.

Cell wall-anchored surface proteins of Gram-positive bacteria, harboring a highly conserved YSIRK/G-S signal peptide (SP_YSIRK+), are deposited at cell division septum and anchored to septal peptidoglycan. The mechanisms supporting YSIRK protein septal trafficking remain elusive. Previously, we identified that LtaS-mediated lipoteichoic acid (LTA) synthesis restricts septal trafficking of YSIRK+ proteins in Staphylococcus aureus. Interestingly, both LtaS and SP_YSIRK+ are cleaved by the signal peptidase SpsB, but the biological implications remain unclear. Here, we show that SpsB is required for cleaving SP_SpA(YSIRK+) of staphylococcal surface protein A (SpA). Depletion of spsB not only diminished SP_SpA processing but also abolished SpA septal localization. The mis-localization is attributed to the cleavage activity of SpsB, as an A37P mutation of SP_SpA that disrupted SpsB cleavage abrogated SpA septal localization. Strikingly, depletion of spsB led to aberrant cell morphology, cell cycle arrest, and daughter cell separation defects. Localization studies showed that SpsB was enriched at the septum of dividing staphylococcal cells. Finally, we show that SpsB spatially regulates LtaS as spsB depletion enriched LtaS at the septum. Collectively, the data suggest a new dual-mechanism model mediated by SpsB: the abundant YSIRK+ proteins are efficiently processed by septal localized SpsB; SpsB cleaves LtaS at the septum, which spatially regulates LtaS activity contributing to YSIRK+ proteins septal trafficking. The study identifies SpsB as a novel and key regulator orchestrating protein secretion, cell cycle, and cell envelope biogenesis.

Importance: Surface proteins containing a YSIRK/G-S-positive signal peptide are widely distributed in Gram-positive bacteria and play essential roles in bacterial pathogenesis. They are highly expressed proteins that are enriched at the septum during cell division. The biogenesis of these proteins is coordinated with cell cycle and LTA synthesis. The current study identified the staphylococcal signal peptidase SpsB as a key determinant in regulating surface protein septal trafficking. Furthermore, this study highlights the novel functions of SpsB in coordinating LtaS-mediated LTA production and regulating staphylococcal cell cycle. As SpsB, YSIRK+ proteins, and LTA synthesis are widely distributed and conserved, the mechanisms identified here may be shared across Gram-positive bacteria.

Structural maintenance of chromosomes (SMC) are ubiquitously distributed proteins involved in chromosome organization. Deletion of smc causes severe growth phenotypes in many organisms. Surprisingly, smc can be deleted in Corynebacterium glutamicum, a member of the Actinomycetota phylum, without any apparent growth phenotype. SMC in C. glutamicum is loaded in a ParB-dependent fashion to the chromosome and functions in replichore cohesion. The unexpected absence of a growth phenotype in the smc mutant prompted us to screen for synthetic interactions within C. glutamicum. We generated a high-density Tn5 library from wild-type and smc-deleted C. glutamicum strains. Transposon sequencing data revealed that the DNA translocase FtsK is essential in an smc-deletion strain. In wild-type cells, FtsK localized to the septa and cell poles, showing polar enrichment during the earlier stages of the life cycle and relocating to the septum in the later stages. However, deletion of smc resulted in an earlier onset of pole-to-septum FtsK relocation, suggesting that prolonged FtsK complex activity is both required and sufficient to compensate for the absence of SMC, thus achieving efficient chromosome segregation in C. glutamicum. Deletion of ParB increases SMC and FtsK mobility. While the change in SMC dynamics aligns with previous data showing ParB's role in SMC loading on DNA, the change in FtsK mobility suggests defects in chromosome segregation. Based on our data, we propose an efficient mechanism for reliable DNA segregation in the absence of replichore arm cohesion in smc mutant cells.IMPORTANCEFaithful DNA segregation is of fundamental importance for life. Bacteria have developed efficient systems to coordinate chromosome compaction, DNA segregation, and cell division. A key factor in DNA compaction is the SMC complex that is found to be essential in many bacteria. In members of the Actinomycetota, smc is dispensable, but the reason for the lack of an smc phenotype in these bacteria remained unclear. We show here that the divisome-associated DNA pump FtsK can compensate for SMC loss and the subsequent loss in correct chromosome organization. In cells with distorted chromosomes, FtsK is recruited and stabilized earlier to the septum, allowing for DNA segregation for a larger part of the cell cycle, until chromosomes are segregated.