Defining the interplay of Klebsiella pneumoniae and host during infection by quantitative proteomics

Question 1

what is k. pneumoniae

Answer 1

• rod shaped, non-flagellated, gram neg, non motile, encapsulated, non sporing, lactose fermenting, faculative anaerobic
• human pathogen affecting thoe with weakened immune system
• causes nosocomial infections: pneumonia, UTI and serious cases intra-abdominal infections
• transmission not known, believed to be direct contact and not through air

Question 2

how is K. pneumoniae treated

Answer 2

• antibiotics
• high prevalence of antibiotic resistant strains, results in a logner treatment regiment so higher probabilty of travelling to other areas of the body

Question 4

what are the majori virulence factors of K pneumoniae

Answer 4

• capsule, lipopolysaccharide, fimbriae and siderophores

Question 5

describe the capsule

Answer 5

• extrapolysaccharide matrix
• composed of strain-specifc capsular polysaccharides referred to as K antigens (K1 through 78)
• protects against phagocytosis, antimicrobial peptides and complement mediated lysis
• prevents activation of early immune response
• acapsular strains tend to be less virulent

Question 6

describe the lipopolysaccaride

Answer 6

• component of the outler leadflet of the cell membrane
• comprised of O antigen, an oligosaccharide core and lipid A
• 9 different O antigens
• protects agaonist complement and antimicrobial peptides
• strong activator of immune response -> use capsule to mask MPS
• plasticity in Lipid A structure aids in avoiding recognition by immune receptors

Question 7

describe the fimbriae

Answer 7

• invovled in adhesion - help to bind to biotic and a biotic surfaces

*involed in biofilm production (bind to catheters and stuff)

K. pneumoniae produced Type 1 and 3

• found in 90% of clincial environmental isolates

Question 8

describe the siderophores

Answer 8

• mol with high iron-chelating properties
• can obtain iron frmot he environment or steal it from host proteins (lactoferrin)
• enterobactin is the most ubiquitous
• counteracted by lipchalin-2, which is produced by humans
• K.pneumoniae expresses several, with ranging binding capabilities

Question 9

classical vs hypervirulent strains

Answer 9

• classical strains cause serious infections such as pneumonia or meningitis when infecting immunocompromised individuals
• hypervirulent strains can affect both healthy and immunocompromised individuals
• the increased virulence is believed to result from resistance to complement and neutrophil activity, increased capsule production and increased sideophore secretion

Question 10

dsecribe the host-pathogen interactions

Answer 10

• pathogen requires resources for replication and survival
• the progression of an ifnection relies on temporally (time) and spatially (location) regualted host-pathogen interactions
• imortant to know howt he pathogen evades host defenses and how the host prevents pathogen survival

Question 11

whats a proteome and a sceretome

Answer 11

proteome = all rptoeins produced

secretome: proteins secreted by bacteria at given time

Question 12

what si rptoeomics

Answer 12

entire protein complement expressed by a genome

• analysis of proteins in a cell tissue or whole organism at a given time under defined conditions
• an organisms genome is more or less constant whereas proteomes differ from cell to cell adn time to time
• the proteome adapts to external or itneral perturbations - therby defining the cells functional sate and determining its phenotypes

Question 13

how is proteomics conducted

Answer 13

• this lab sues mass sppec
• good to dientify proteins

Bottom up proteimics: proteins are digested into peptides prior to analysis

Top down: looks at whole protein

Question 14

how are preotins prepared for testing

Answer 14

1. collect sample and extract them
2. digest using lysine or trypsin, so have little peptides
3. purification to get rid of salts and contaminants
4. separate samples by HPLC
5. Identify peptides by mass spec
6. process samples by MaxQuant
7. perseus: statistical analysis

Question 15

hwo does metal ion avaiabilty affect proteome of K.pneumonea? why did they look at iron

Answer 15

*care bc of nutritonal immunity: host will sequester free metal to restrict growth - bacteria has methods to counteract this

• looked at iron bc one of most studied emtal ions, important roles in DNA replication and oxygen metabolism
• siderophores are primarily secreted with goal of acquiring iron
• iron availablity affect capsule biosysntheis

Question 16

what does volcano plot show

Answer 16

• look at all proteins and look at different levels of expression - comate limited iron to the diff concentration
• point outside the asympotoes are statisitcally significant
• everything in red is more abundant in limited condition

Question 18

what is Lon protease

Answer 18

• serine pepidase

role in cellular homeostasis

• degredation of mutant and abnormal protein

*study found that it is highly epxressed in secretome, identified putative extracellular role in iron homeostasis

• get this elongated phenotype

Question 19

main findings of iron on proteome for K.pneumoniae

Answer 19

• identified novel role for lon protease in iron homesostaties
• defined the impact of iron availabiltiy on the proteome and secretome of K.pneumoniae
• future studied could aim to identify Lon degradation targets and characterize their role in iron homeostatis

Question 20

what is known about K.pneumoniae host pathogen interaction

Answer 20

• most genomic approached
• he looks at proteins produced when bacteria is gorwn with marophages then uses in silico analysis to find what proteins are expressed

Question 21

how are mutants made

Answer 21

• use homologous recombination
• have risistance marker and clone with voerhangs, amplify with Pcr and voerhangs match gene of interest
• use lmbda red recombination and swaps out gene with resistance marker
• only that bacteria can then continue to grow bc has resistance

Question 22

descibre murine infection using kncokout strains

Answer 22

• compare the wild type strain with knock out lines
• compare health and survival of mice, collect organs for bactieral count (how is is bactiera surviving in host, how does mutation effect bacterias survial), look at blood to cytokines

Question 23

how is immunofluorescence used

Answer 23

• good wat to look at rpoteins that are secreted and interact with host
• have protein of interest and produce it with a tag
• antibody binds to antigen and then secondary antibody bidns to that which has a fluorophose
• can use fluroescent microscopy to visualize protein of interest

Question 24

describe co-immunoprecipitation sample prep

Answer 24

• express system in klebsiella cells and while thats happening the protein of interest is binding to host protein and bacteria protein
• use antiflag antibody to purify target interactions - then use proteomics to find what protein of interest is binding to and interacting with