The outer membrane (OM) of P. aeruginosa is a semi-permeable barrier that contributes to antibiotic resistance by reducing uptake. Finding strategies to circumvent this barrier is a major challenge. One approach involves screening in physiologically relevant conditions to identify novel activity in existing molecules. We discovered that thiostrepton (TS), a thiopeptide antibiotic with no reported activity against Gram-negative bacteria, hijacks the pyoverdine siderophore transporters FpvA and FpvB to cross the OM under iron limitation to inhibit translation. Using TS, we subsequently showed that FpvB is not primarily a pyoverdine transporter, but rather a promiscuous transporter for siderophores ferrichrome and ferrioxamine B. Our work with TS suggested that other thiopeptides may use siderophore transporters for entry into the cell. This hypothesis led to a screen to identify other thiopeptides with activity against P. aeruginosa, uncovering two other thiopeptides, thiocillin and micrococcin, that use the ferrioxamine transporter FoxA for uptake. We discovered another siderophore, bisucaberin, could also use FoxA for uptake and our collaborators solved the crystal structure of bisucaberin bound to FoxA. Through biochemical approaches, we characterized how FoxA accommodates structurally distinct ligands. Finally, we screened known large natural product antibiotics with no pseudomonal activity under nutrient limitation and discovered that the glycopeptide vancomycin inhibits growth by blocking peptidoglycan crosslinking. This pilot screen emphasizes the importance of screening for antibiotics under physiologically relevant conditions to avoid overlooking potential hits. Overall, the findings from these studies can be used to guide medicinal chemistry efforts to develop novel siderophore-antibiotic conjugates for the treatment of P. aeruginosa infections. These results also help us gain a deeper understanding of the mechanism of binding and uptake through siderophore transporters and the range of substrates that can be taken up. / Dissertation / Doctor of Philosophy (PhD) / Antibiotic resistance is a growing crisis that threatens modern medicine, and it is becoming more challenging to discover truly new antibiotics to combat this threat. Intrinsic resistance conferred by the outer membrane of Gram-negative bacteria restricts the entry of many antibiotics, especially larger antibiotics that would otherwise inhibit the growth of Gram-positive bacteria. Consequently, there are fewer treatment options for infections caused by Gram-negative bacteria and developing new antibiotics that can cross the outer membrane remains a significant challenge in drug discovery. My work describes the discovery of a class of antibiotics that can bypass the outer membrane using specific outer-membrane nutrient transporters. Using biochemical, structural biology, fluorescence microscopy, and molecular biology techniques, we uncover the molecular determinants of uptake of these antibiotics for their respective transporters. These results can inform the design of novel narrow-spectrum antibiotics that can overcome the outer membrane barrier to combat antimicrobial resistance.

Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa(Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S-and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ~10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions.

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The primary mode of growth for bacteria in the environment and during infection is as a biofilm–multicellular assemblages encased in a self-produced matrix. Bacteria growing in biofilms must contend with the difficulties of resource limitation and competition in order to reap the benefits of increased protection from external stresses including the antibiotics used against them. With the rise in multi-drug resistance, understanding the interplay of the complicated processes that make this growth style possible will help us develop better treatment options. Cells must maintain redox homeostasis in order to carry out metabolism and avoid death. In the pathogen Pseudomonas aeruginosa, oxygen is the preferred terminal electron acceptor used for this purpose. However, oxygen is often scarce under natural growth conditions, where opposing rates of diffusion and consumption lead to the formation of steep gradients. Under conditions of oxygen limitation, the metabolically versatile P. aeruginosa can use two major redox balancing strategies: (i) denitrification (i.e., respiration of exogenous nitrate) and (ii) reduction of endogenous redox-active pigments called phenazines. The work in this thesis describes novel regulatory mechanisms for redox homeostasis with an emphasis on the biofilm lifestyle. Chapter 1 will introduce the necessary background about redox pathways and homeostasis in P. aeruginosa, and how this organism senses chemical cues and transduces this information into physiological adjustments that support metabolic activity and survival. Chapter 2 highlights the remarkable versatility of P. aeruginosa in producing multiple terminal oxidases, particularly the cbb3-type terminal oxidases encoded by partially redundant operons, which have the potential to generate 16 isoforms. The interaction between small-molecule virulence factors, such as cyanide, and the respiratory chain adds complexity to this system. This study uncovers the regulatory role of MpaR, a predicted pyridoxal phosphate-binding transcription factor, in governing expression of a cbb3-type terminal oxidase subunit in response to endogenous cyanide. Chapter 3 demonstrates that pyocyanin, a terminal phenazine product, promotes metabolic activity at a depth in biofilms. However, production of pyocyanin and precursor products is stressful to cells particularly when electron donors are limiting. This work presents the global regulators RpoS and Hfq/Crc as regulators of phenazine production to balance toxicity and metabolic support. Finally, Chapter 4 identifies the first member of the P. aeruginosa phosphotransferase system, PtsP, as an oxygen-independent regulator of phenazine production and denitrification. The research presented in this thesis sheds light on P. aeruginosa’s adaptive tactics for thriving under adverse conditions. Understanding the physiology of this bacterium under conditions relevant to biofilm-based infection provides insights into its strategies for long-term colonization in host environments and opens the door for development of more effective antimicrobials in the face of a world-wide antibiotic crisis.

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