P. aeruginosa Guest Lecture Flashcards
ESKAPE pathogens (2)
- list of bacteria that new antibiotics are urgently needed for
- prevalent causes of hospital-acquired infections
how do ESKAPE pathogens current evade antibiotics (4)
- antimicrobial inactivation
- persistence
- bacterial target site modification
- reduced antibiotic accumulation
what is an example of antimicrobial inactivation
- β-lactam modification
what is an example of persistence
- biofilm formation
what is an example of bacterial target site modification
- cell wall alterations
what is an example of reduced antibiotic accumulation (2)
- porin alterations
- efflux pumps
P. aeruginosa (3)
- gram negative
- facultative anaerobe
- opportunistic
opportunistic pathogens
- preferentially infects immune-suppressed or compromised individuals
what niches do P. aeruginosa infect
- infect many different bodily niches
what antibiotics do we depend on to manage P. aeruginosa infections (4)
- macrolides
- aminoglycosides
- quinolones
- β-lactams
macrolides
- target 50s ribosomal subunit
aminoglycosides
- target 30S ribosomal subunit
quinolones
- target DNA gyrase and topoisomerase IV
β-lactam
- inhibit peptidoglycan cross-linking
what factors can lead to resistance to antimicrobials (3)
- intrinsic features of the bacteria
- acquired features
- adaptive lifestyles
how can P. aeruginosa exhibit an adaptive lifestyle in antibiotic resistance (2)
- swarming motility
- biofilm formation
swarming motility (2)
- describe how P. aeruginosa exhibit rapid surface motility
- causes groups of cells to raft together and propel themselves over surfaces
swarming motility: what is used to raft cells together
- type IV pili
swarming motility: what is used to propel over surfaces
- flagella
how is swarming motility initiated (2)
- RhlRI quorum sensing signaling
- signaling begins once bacteria reach critical cellular density
RhIRI quorum sensing
- regulates production of rhamnolipids
what is the role of rhamnolipids
- glycolipid biosurfactants that reduce surface tension of mediums
what produces rhamnolipids
- produced almost exclusively by Pseudomonads
what are rhamnolipids composed of (2)
- one or two rhamnose moieties
- fatty acid chains
biofilm formation (2)
- groups of cells adhere to a surface and form structured bacterial communities in extracellular matrixes
- downregulation of metabolic processes
extracellular matrix composition
- required (3)
- conditional (1)
- always consists of polysaccharides, extracellular DNA, alginate (glycoprotein)
- rhamnolipids only required for structured biofilm formation
reverse genetics
- study of phenotypic effects caused by a genetic knock-out
what tools can be used to perform reverse genetics in parallel (2)
- transposon mutagenesis
- T-Seq
how did the lab identify regulative genes needed for adaptive antimicrobial resistance (3)
- created transposon library
- subjected the pools of mutants to different conditions, such as swarming or biofilm states
- used T-Seq to map and determine read counts for each insertion site
analyzing read counts: severe defect
- mutant is not detectable in experimental group
analyzing read counts: decreased fitness
- decreased mutant abundance in experimental group
analyzing read counts: increased fitness
- increased mutant abundance in experimental group
how were genes involved in motility and adaptation affected in swarming bacteria
- genes were enriched
what were adaptive growth states regulated by
- two-component systems, which were over-represented in data
two-component system role
- regulate bacterial virulence and immunogenicity
two-component system composition (2)
- membrane bound sensor kinase (SK)
- cytoplasmic response regulator (RR)
two-component systems: membrane bound SK (3)
- binds periplasmic signal
- undergoes conformational change
- kinase activity
two-component systems: cytoplasmic RR (2)
- most are transcriptional regulators
- induce or inhibit expression of genes in response to stimulus
what two-component system was detected in each Tn-Seq dataset
- NtrBC
NtrBC (2)
- non-canonical TCS
- no known periplasmic signal
when is NtrBC activated
- when intracellular glutamine is low
NtrC
- bacterial enhancer binding protein of RpoN in the NtrBC TCS
what was the research hypothesis
- if NtrBC signaling is important for adaptation in cell culture and animal mode of disease, P. aeruginosa ntrB, ntrC, and ntrBC mutants will be defective for adaptive phenotypes in vitro and in vivo
research aim 1
- screen P. aeruginosa WT and mutant ntrB, ntrC, and ntrBC for adaptive phenotypes/antimicrobial resistance in vitro
what was the finding from research aim 1
- delection of ntrB, ntrC, and ntrC reduced swarming due to rhamnolipid production
research aim 2
- screen P. aeruginosa pathogenesis in WT and mutant ntrB, ntrC, and ntrBC in skin infection models
what was the finding from research aim 2 (2)
- NtrBC regulated invasiveness and virulence during skin infection
- NtrBC was important for ciprofloxacin resistance during abscess infection
research aim 3
- establish a sinusitis infection model to determine tissue-specific effects of NtrBC on adaptive regulation in vivo
what were the findings from research aim 3
- NtrBC regulation of colonization was observed in sinuses, but not skin of mice, suggesting tissue-dependent regulation by NtrBC
research aim 4
- examine host-pathogen and interspecies interactions of P.aeruginosa WT, and mutant ntrB, ntrC and ntrBC in vitro
virulence
- ability to inflict damage on a host
P. aeruginosa virulence factors (3)
- siderophores
- bacterial appendages
- secreted molecules (exotoxins, proteases, etc)
what were the findings from research aim 4 (4)
- NtrBC selectively impacted on P. aeruginosa interactions with host cells
- NtrBC regulated virulence factor production in vitro
- most infections of skin and respiratory tract are polymicrobial, which impacts patient prognosis and antimicrobial susceptibility of species
- NtrBC confers competitive advantage on P. aeruginosa in vitro and in vivo
research aim 5
- define transcriptional profiles of WT and mutant ntrB, ntrC, and ntrBC than compare them to transcriptional profile of mutant rpoN
what were the findings from research aim 5 (2)
- NtrB and NtrB had distinct, but overlapping impacts on transcriptome
- NtrBC and RpoN impact transcriptome of P. aeruginosa more than NtrB or NtrC
what were the conclusions from the study (2)
- NtrBC regulates adaptive phenotypes and intercellular interactions of P. aeruginosa in vitro and in vivo
- regulatory activity of NtrBC could be partially independent of RpoN
how can the conclusions from the research be utilized (2)
- NtrBC could be targeted to add value to existing treatment options
- emerging therapies that target NtrBC should be screened for activity in host-like conditions to maximize potential for clinical translation
