Download Pseudomonas aeruginosa lytic bacteriophage oKMV

Pseudomonas aeruginosa lytic bacteriophage oKMV receptor analysis.
Andrew Chibeu
Samenvatting
van
het
onderzoek
/
Summary
of
Research
Viruses
that
specifically
infect
bacteria
(bacteriophages
or
phages)
are
obligate parasites and ubiquitous in nature. Since their discovery in the
early twentieth century, research into the biological nature of phages has
contributed
immensely
to
their
exploitation
in
providing
biotechnology
tools.
In the recent past, there has been renewed interest in bacteriophages as
potential
antibacterial
agents
against
the
increasing
number
of
antibiotic-resistant bacteria. Among the bacteria for which the potential of
such phage therapy has found application is Pseudomonas aeruginosa, an
opportunistic pathogen whose infections occur in patients with compromised
host defences such as those with HIV, burn patients or with cystic fibrosis.
These
infections
are
difficult
to
treat
with
conventional
antibiotic
therapies
and
often
result
in
morbidity
and
mortality.
As the main drawback of using phages in treatment of bacterial infections is
their narrow host range, there is the need to develop cocktails containing
phages that lyse a wider variety of host strains. Any phage cocktail should
contain phages that use a diversity of receptors, to ensure an expanded host
range and also to suppress the emergence of phage-resistant strains. Hence,
phage receptor studies are paramount where phage therapy is envisaged.
BacteriophagephiKMV is a virulent phage of P. aeruginosa isolated from a
water source in the Moscow suburbs. Its genome sequence and basic biology
have been the focus of active research at the K.U. Leuven Laboratory of Gene
Technology (LoGT) in the past decade. To date, the receptor and host range
determinants of phage phiKMV had not been determined. This dissertation
focuses on determining the receptor of bacteriophage phiKMV and developing
tools for the identification of other virulent phage receptors for phage
therapy
applications.
Host infectivity screens, adsorption tests as well as the classical mutant
characterization
approach
were
applied
in
identifying
host
receptor
proteins/structures.
These
tests
established
that
the
growth
of
P.
aeruginosa
strains
on
solid
surface
was
essential
for
phage
phiKMV
infectivity. As follows, the receptor structures are accessible to the phage
when
expressed
under
these
growth
conditions.
These
structures
were
identified to be type IV pili through functional complementation of the
isolated spontaneous phage phiKMV-resistant mutant strain with the genomic
library
of
the
wild
type
phiKMV-sensitive
strain.
Further
in
depth
analysis
and
characterization
of
the
spontaneous
phiKMV-resistant mutant strain established that the phage phiKMV resistance
mechanism
is
a
complex
process,
possibly
involving
post-transcriptional
regulation
of
the
type
IV
pili
biogenesis
genes.
A second putative host receptor protein (PA4736) was identified by a library
scale yeast two-hybrid screen. To our knowledge, this study was the first
time the yeast two-hybrid system was used in determining phage-host receptor
interactions. This P. aeruginosa PAO1 protein was found to interact with
phage phiKMV gp48. PA4736 is co-transcribed with the outer membrane protein
PA4735 and it is highly likely that these proteins interact. It is thus
proposed that the P. aeruginosa protein PA4736, which had no previously
annotated function, is the secondary receptor for phage phiKMV. When
successfully verified by an in vitro technique, the interaction between
phiKMV gp48 and P. aeruginosa PAO1 protein PA4736 will be ascertained to be
a true positive interaction. Recombinant expression of the proteins followed
by in vitro interaction assays would provide data which when combined with
that from this study would lead to the functional annotation of the phage
phiKMV
co-receptor
protein,
PA4736.
Type IV pili-dependent infectivity of phage phiKMV has profound implications
in its use for phage therapy applications, such as its potential influence
when the need for phage propagation in batch cultures arises. The role of
the
differential
expression
the
type
IV
pili
biogenesis
genes
in
bacteriophage phiKMV-sensitivity would make the phage a valuable tool in the
typing of P. aeruginosa strains with the intact type IV pili assembly
complex. Hence, the phage can assume a role in experiments to determine the
presence or absence of intact type IV pili assembly machinery and wild type
twitching motility. Several tools for use in phage receptor studies were
developed in the course of this research. These tools can be used in
studies to identify other P. aeruginosa phage receptors for phage therapy
applications.