KU BIOL 506 exam 2

Why is cloning and expressing random DNA fragments helpful in identifying virulence factors?

-Cloning helps identify a mutant that is now virulent

How is cloning and expressing the random pathogenic DNA fragments in avirulent hosts done?

-Positive selection (gain of function)

-Transcriptional fusion: identifies conditions where virulence factors are expressed

-Reporter genes like LacZ

-fluorescent proteins

-Bacterial luciferase

-Transposon mutagenesis

How does transposon mutagenesis aid in identification of bacterial virulence factors?

-Random insertions of transposons are used as insertion mutations into a pathogenic genome and screened for loss of virulence

-MUST have a selectable marker.

How is TN-Seq used in disease models?

-It is able to identify essential genes from growth in non-selective rich lab media; also identifies virulence genes from growth in selective media

-In vitro: helps determine precise location of where each transposon landed

What are some setbacks of TN-seq?

pools complementation of secreted factors such as toxins and proteases

What are some limitations of transposon mutant studies?

-some carry termination sequences early on

-polarity

-will only target non-essential genes and may very by experimental parameters

-Functionality redundancy may miss important protein families

Why use transcriptional reporters (gene fusions)?

-Overcomes setbacks

-helps find mutants in a wide variety of environmental conditions

-helps identify genes encoding regulatory proteins that control expression of virulence genes

How can RNA Seq be used to identify transcriptional profile differences between strains?

It can use Next Gen sequencing to determine what bacterial genes are expressed during infections

-Examines transcriptional factor mutants to detect RNA differences and find regulons

-Isolate RNA and convert to stable cDNA for sequencing

-Tells quantitative differences between transcriptional abundance

What is IVIAT?

Antibody based genomic method that can be used to identify genes induced during human infections, but can avoid the use of animal infection models

How does IVIAT work?

1. collect infected patient sera

2. sera is pooled with pathogen cells grown in vitro (leaving only cells expressed in vivo)

3. expression is induced (sera will only contain in vivo cells which will react with proteins)

How do you identify candidates for a vaccine?

2 main groups

1. Polyvalent vaccines - serogroups A,C, and Y; effective for everyone 2+ years old

2. Serogroup B - weak immunogenic and needs acquired immunity from cross-reacted antigens (working on improvments)

How do you identify host factors that are important in disease resistance and pathogenesis?

Use RNAi or CRISPR-Cas9

Identifying using RNAi

-allows for screening of large libraires of host genes required for bacterial pathogenesis

-siRNA induces selective cell-mediated degradation of any mRNA that has been homology to the siRNA, resulting in gene silencing

-screens thousands of genes simultaneously

Identifying using CRISPR-Cas9

-allows for fast and precise manipulation of host genomes to study gene function

-Identifies distinct host cell pathways that confer susceptibility to infection

-Type III secretion system in Vibrio

What are some reasons pathogens cannot survive outside of the cell for very long?

Threats including

-being eaten

-exposure to chemicals

-lack of nutrients

-sunlight

-lack adherence sites

What are some survival tactics of bacteria living outside a host?

-Endospores

-desiccation tolerance (limit processes that dehydrate or deplete nutrients)

-Secondary metabolites

-efflux pumps

-metabolic diversity

-biofilms

What is a siderophore?

-an iron binding protein that is used to capture iron from the environment/host

-Uses 2 primary structure: catechol and hydroxamates

If a bacteria does not have its own siderophore, how does it get some?

-takes some from other bacteria

-steal (freeloading basically)

-produce toxins

What are the 5 host factors that sequester iron?

-Lactoferrin

-Transferrin

-Ferritin

-Heme

-Siderocalin

What is an example of non-fimbria adhesions?

-S. pyogenes

-F protein binds to fibronectin

-M protein: alpha helical structure that resembles pili

-Binds H factors and destroys C3 convertase to avoid phagocytosis and complement pathway