This document summarizes research on sortase enzymes. Sortases are bacterial transpeptidase enzymes that covalently attach secreted proteins to the bacterial cell wall. They play important roles in virulence and pathogenesis. The document discusses various classes of sortases, their mechanisms of action, roles in antibiotic resistance and biofilm formation. It also outlines applications of sortases in areas like vaccine development, drug targeting, and protein immobilization. Overall, the document provides an overview of the sortase enzyme family and their significance in microbial physiology and disease.

1. Dr. G.KALAISELVI
DPV(M)17002(ABT)
PART TIME Ph.D SCHOLAR
DEPARTMENT OF ANIMAL BIOTECHNOLOGY
MADRAS VETERINARY COLLEGE, CHENNAI-7
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SORTASE ENZYME FAMILY AND ITS IMPORTANT

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3. ANTIBIOTIC RESISTANCE
3
Transposons
Genetic
mutation of
bacteria
Horizontal
transfer of
resistance
genes
between
bacteria.
Plasmids
several
resistance
genes giving
resistance
Improper usage of antibiotics – accumulation in meat,
milk, egg and soil
Persistence of antibiotic resistance
Development of superbug -MRSA

4. ANTIBIOTIC TARGET AND
RESISTANCE EVALUATION
4

5. SORTASE ENZYME - A VIEW
5
The Peptidoglycan
glycan polymers
made up of
repeating chains of
N-acetyl
glucosamine and N-
acetyl muramic acid
Peptide stem composed
of 5 aminoacid at 3
position- L-Ala-D-iGlu-
L-Lys-DAla- D-Ala
with a pentaglycine
inter-peptide branching
off the L-Lys.
(Schneewind et.al.,
1992)

6. SORTASE ENZYME - A VIEW
6
Sortases are Gram-
positive bacterial
extracellular
transpeptidase enzymes -
covalently attaching
secreted proteins to the
peptidoglycan cell wall.
(Schneewind et.al., 1992)
Schneewind
(Staphylococcus
aureus in 1999
named as
sortase A (SrtA)-
Sort tagging
protein.

7. FUNCTIONS OF CELL WALL PROTEINS
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8. ROLE OF SURFACE PROTEIN
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9. MECHANISM OF ACTION OF SORTASE ENZYME
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Proteins destined for cell wall anchoring are first initiated into the Sec pathway by an N-terminal signal
peptide (SP).
Cleavage of the LPXTG motif, a thioester-linked acyl-enzyme intermediate is formed between sortase
and C-terminal threonine of surface protein.
Resolution of the acyl-enzyme occurs through nucleophilic attack of the amino group of the pentaglycine
of lipid II to generate lipid II-linked surface protein.
The black bar represent the hydrophobic domain and positively charged tail of cell wall sorting signals
X indicates potential points of inhibition in the “sorting pathway.”

10. MICROBIAL SURFACE PROTEIN
10
Microbial Surface Components Recognizing Adhesive Matrix
Molecules (MSCRAMMs ) (Mazmanian et.al., 2001)
Clostridium
difficile
SPXTG or
PPXTG
instead of
LPXTG

11. SURFACE PROTEIN VIRULENCE
11
(Dramsi et.al.,2005)
Surface displaying virulence factors - drug targets
Molecular basis of substrate recognition.
Catalysis —binding of the sorting signal to the active site
Robust biochemical assays to monitor pilus assembly and novel
substrate analog to visualize nucleophile recognition (Schneewind
et.al.,1993)

12. Sortase enzyme classes
12

13. FUNCTIONS OF SORTASE -1
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Cell wall formation and display of surface protein
Construction of pili, flagella – Bacterial adhesion and conjugation
Development of virulence, infection and colonization of bacteria-
antibiotic resistance and immuno evasion and bacterial spore
formation(Danne et.al 2012)

14. IMMUNO EVASION MECHANISM
14

15. MULTI DRUG RESISTANCE S.AURIEUS
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PCR amplification of the srtA gene
Primers detail
Forward primer ===5'-AAACCACATATCGATAATTATC-3'
Reverse primer === 5'-TTATTTGACTTCTGTAGCTACAA-3‘
PCR product was cloned into pBAD/Thio-TOPOvector and transformed into E.
coli TOP10.
The sequence of the S. aureus ATCC 6538P srtA clone showed 99%identity at the
amino acid level with other S. aureus

17. APPLICATIONS OF SORTASE -1
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Biofilm vaccine,
capsular and
flagellar vaccine
Mutagenic
strains -
industrial
application
Direct couple the
drug–linker to
antibodies
Lactobacillus spp.
intestinal mucosal
adhesion (Roos
et.al.,2002)
Sortase inhibitor compounds -controlling multi
drug resistance bacterial species control
(Schneider et.al.,2004)

18. APPLICATIONS OF SORTASE -1
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Inhibition of sortase
enzyme -antibacterial
drugs in case of MDRS
Development of new
generation of antibiotic
against very virulent
bacterial species

19. Targeting haem receptor
Sortase B enzymes are responsible for sorting a single
substrate (IsdC) bacterial species grown under iron
deprived condition
S. aureus and B. anthracis a single class sortase B enzyme
anchors the IsdC haemoprotein to the cell wall.
 IsdC is to facilitate haem capture from human
haemoglobin by relaying haem from upstream haem-
receptors to a membrane transporter complex that imports
haem into the cell (Maresso et.al., 2008)
19

20. ROLE OF SORTASE IN LACTOBACILLUS INTESTINAL
MUCOSAL ADHESION
20
Anchoring
machinery in
Lactobacillus
development
of oral
vaccines
Display of cell
surface
proteins-
probiotic
microbes
associated with
the intestinal
mucosa.
Bacterial -host interactions.- Antigen display in
intestinal mucosa (Dieye et.al.,2010)

21. LACTIC ACID BACTERIA IN MUCOSAL DISPLAY
21
Protein targeting and Protein attachment
Secretary (Sec) pathway or twin-arginine
translocation (TAT) pathway
Secretary
pathway unfolded
protein targets
The TAT
pathway
helps to
transport
folded protein
to the cell’s
exterior

22. Industrial application
22

23. Sortagging principles
23
206-amino acid protein with
a potential N-terminal
signal peptide & cysteine at
position 184
Protease
activity
Transpeptidase
activity

24. SOLID SURFACE SYNTHESIS
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Sortase-mediated ligation -Sortagging
Protein - protein interaction, Lipidation,
PEGylation, protein immobilization for drug
design and protein-modification.

25. INDUSTRIAL APPLICATION –SOILD SURFACE
DISPLAY
25
The conjugation of two or
more proteins in solid surface
SORTAGGING -
immobilization of proteins on
a surface, labeling of proteins
for immune diagnostics.
Engineered sortase
substrate -140 times higher
activity –proteins entirely
chemically synthesized
Drug development and
target delivery of
therapeutic substances

26. SORTASE MEDIATED ANTIBODY
LABELLING
26
Sortase mediated
biotin labelling
Sortase mediated
fluorescent labelling
Antibody sequencing
Cell based ELISA
Therapeutic Mabs and
viral vaccine

27. Multifunctionalized protein nano particles
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28. Current research
associated with sortase
enzymes
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Chronic mastitis -bacterial
growth in the form of adhesive
colonies surrounded by a large
exopolysaccharide matrix
creating a biofilm (Costerton et
al., 1999).
Biofilms are resistant to
macrophage mediated
phagocytosis due to their
aggregate size and also become
resistant to some antibiotics
(Monzon et al., 2002).
Development of biofilm
vaccine – control of chronic
mastitis

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 Acceptor lipopeptide (AcLP).
acceptor oligoglycine and
hexadecanoic acid with or without
conjugated spacer (PEG) AcLP
was incubated with DPPC-Chol-
liposome
Donor lipopeptide with donor
sequence of SrtA (LPETG) and
hexadecanoic acid with or without
conjugated spacer (PEG) AcLP
was incubated with DPPC-Chol-
liposome

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Chemicals:
Methyl(2E)-2,3-bis(4-methoxyphenyl)prop-2-enoate, diaryacrylonitriles and (Z)-3-
(2,5-di methoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA
Maltol-3-O-(4′-O-cis-p-cumaroyl-6′-O-(3-hydroxy-3-methylglu-taroyl)-β-
glucopyranoside, bis(indole)
Natural products:
Alkaloids, flavonoids, substrate-derived peptides, such as vinyl sulfone
peptidomimetics and threonine analogues used as potent sortase inhibitors

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37. 37
Cys205 attacks the peptide bond
between threonine and glycine.
SrtA crystal structures ofS. mutans
(blue) and S. aureus (magentas) with its
inhibitor (green).
The colours of carbon, hydrogen, oxygen, nitrogen and
sulfur atoms of the inhibitor are cyan, white, red, blue
and orange, respectively.
The colours of carbon, hydrogen, oxygen,
nitrogen andsulfur atoms of the inhibitor are
cyan, white, red, blue and orange,
respectively.

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40. Advantages of engineered RBCs
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Target drug delivery
Matured RBCs doesn’t
carries nuclei
possibility of
tumerogenesity less –
replaces stem cell risk.
Life span of RBCs 120
days – present micro
and macro circulation
Engineered RBCs with
bioavailable
therapeutics –
prolonged availability

41. 41
Rational design of substrate
mimetic inhibitors based on the
structure of the enzyme and
enzyme substrates,
identification of novel inhibitors
among natural products.
Discovery development of
SrtA inhibitors via high-
throughput and in
silico screening of small
molecule libraries followed by
structural optimization.

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43. Functionalized magnetic nano particles
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45. CONCLUSION
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Sortase enzyme super family ---- play key
roles in microbial physiology,
pathogenesis and virulence development
of bacteria.
Whole genome sequencing efforts have
identified nearly a thousand sortase
homologues whose functions are only
discovered.
All sortases characterized based on
cysteine transpeptidases, protease and
ligase activity further functions of sortase
will be explored in future.
Vaccine development and
immunodiagnostic and bio therapeutic
approaches should be explored

46. FUTURISM OF SORTASE
ENZYME RESEARCH
46
 Further Structure -function
studies needed to identify enzyme
determinants that control substrate
specificity.
 Applications of sortagging
activity of sortase enzymes to be an
extremely useful and versatile
molecular biology tool but very
minimum research work carried out
in related to sortase enzymes in
world wide
Antibiotic resistance threats has
to be focused more in India but
not much research work yet to be
carried related sortase enzyme

47. PATHOGENS
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