Bacterial Neuraminidase

Rosemary CullanderCornell Leadership Program

for Veterinary Sudents- 2012 Scholar

Neu5AcSialic Acid (C9) OR

Neuraminic Acid

Function: Eukaryotic cell

• stabilise glycoconjugates

• mediate cell-cell regulationo chemical messengers

• regulate transmembrane receptor function

• affect membrane transport

• control t1/2 of circulating glycoproteins

• contribute to permselectivity of glomerular endothelium

Function: Bacterial cell

• Carbon + Nitrogen sourceo hexosamine=

ManNAco Pyruvate

PEP Acetyl CoA

• reason for sialic acid structural diversity in bacteria + hosts uncertaino bacteral defense?o conserve water?

Bacterial Neuraminidase OR Sialidases

• glycosylhydrolases that cleave sialic acido extracellular- NanH

S. enteritica Arizonae

• Functiono increases local concentration of free sialic acid that can then be

utilised by bacteria ex) mucus glycoprotein cell surface biosynthesis (E. Coli K1/ S. agalagtiae capsule) food source (catabolism) ex) E. coli K12 signalling molecule- sialic acid or metabolic product

• direct interaction with sialic acid specific lectins (Siglec family)

• CD24 (glycoprotein)-Siglec10 (IG-like lectin that recognises SAcid) o mutation= increase inflammationo protective function depends on CD24 sialyationo disrupting pattern-recognition= decrease host response

to infection immune evasion (anti-recognition)

http://en.wikipedia.org/wiki/Bacterial_neuraminidase

http://mic.sgmjournals.org/content/153/9/2817.fullSialic acid utilization by bacterial pathogens- E. Severi et al

Sialic AcidAcquisitio

n(nan

operon)

http://2009.igem.org/Team:NYMU-Taipei/Project/Receptor/Sialic_Acid

Nag AB

NanA

Nan EK

Neu CB

Neu A

Catabolism• NanA (sialidase)• Nan EK• Nag AB

Biosynthesis + Sialylation• NeuCB• Neu A

Scavenging• NanH (sialidase) of S.

enteritica Arizonae (50%)

Signaling (free S.A.)1. binding of S.A. to

sensor-kinase2. intracellular transport

and binding to regulator (aka nanR)

NanH

Genetic organization of E. coli nan pathway of sialate catabolism.

Kalivoda K A et al. J. Bacteriol. 2003;185:4806-4815

Aldolasae

Nan (N-acyl neuraminate) operon

SIALIC ACIDN-acetylneuraminic acid

derepressed if grown on sialic acid-only medium

2ndary transporter

Genetic Organization of E. coli nan-pathway of sialate catabolism

Nan R regulation and type 1 fimbriae E. coli K-12

• FimB- only? recombinase able to switch cell from afimbriate state to fimbriate

-able to switch (fimAICDFGH) on <--> off

• FimB= pathogenic factor in Crohn's Disease, E. coli invasion

• yjhATS and FimB include operator with 20/23 similar nucelotide sequence with nanRo appears to regulate FimB expression from a

distance

• exogenous sialic acid reduced FimB expression at normal urine concentrations (physiologic role?)

• NanR acts as:o classical nan repressoro antirepressor regulating FimB expressiono repressor of yjhATS and yjhBC

• Precise binding of NanR to nanA operator unknown

http://2008.igem.org/Team:NYMU-Taipei/Project/Attachment

Current Neuraminidase Inhibitors

Laboratory Available

• Siastatin

• 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acido Neu5Ac2en

• 2,3-dehdro-2-deoxy-N-glycolyl-neuraminic acido NeuGc2en

Neuraminidases- on the market

• Oseltamavir

• Permavir

• Zanamivir

http://www.virology.ws/2009/12/23/influenza-neuraminidase-inhibitors-work/

Current Project Goals1) In silico- find bacteria in collection that may express EXTRACELLULAR neuraminidase- nanH/ operon ex) e.coli, S. enterica

2) Neuraminidase activity reporting: (extracellular breakdown)- look at gene expression (RT pcr/qpcr), compare baseline/ negative control (bug w/no gene)- if have gene but no expression- set up studies for different sialic acid concentrations to see if inducible

a) Qpcr at different time points/ concentrations to look for neuraminidase expressionb) OR chromagen/ flourescence studies on bacterial cultures

3) test function with neuraminidase inhibitors: Function of gene when expressed/ not expressed- cell culture system- add sialic acid

4) Obtain parvo samples for bugs (E. coli) that may be expressing exogenous neuraminidases/ other virulence factors, look at sialic acid concentrations- determine if invasion is sialic acid dependent?