| Summary: | Memoria para optar al Título de Bioquímico === The genus Salmonella belongs to the Enterobacteriaceae family and comprises
two species: S. bongori and S. enterica. Both species include more than 2500
serovars. Within S. enterica serovars, the most studied are S. Typhi,
S. Typhimurium and S. Enteritidis, the latter being the main causative agent of
salmonellosis worldwide.
During its infective cycle, Salmonella needs to adapt to variations in different
environmental conditions, such as changes in pH, osmolarity and oxygen
availability. In addition, these conditions serve as environmental signals that
modulate the expression of virulence factors. One of these factors is the
lipopolysaccharide (LPS), the main component of the envelope of Gram negative
bacteria.
The LPS presents three structural domains: the O antigen (AgO), the core region
and lipid A, also called endotoxin. The latter anchors the LPS to the outer
membrane, and is a known virulence factor responsible for the induction of the
immune response. The lipid A domain is capable of undergoing covalent
modifications, which modulate its toxicity and helps bacteria to evade antimicrobial
agents and the immune system of the host.
One of the main environmental cues faced by Salmonella during its infectious cycle
is the oxygen availability. During the aerobic-anaerobic transition participates the
global regulators ArcA and Fnr. Results from our laboratory show that changes in
the polymerization degree of the AgO in response to oxygen availability is
modulated by these regulators. This observation led us to examine whether global
regulators Fnr and ArcA are involved in the control of probable oxygen-dependent
modifications of lipid A in response to oxygen availability.
The main goal of this study was to determine the effect of oxygen availability on
covalent modifications of lipid A in S. Enteritidis and the participation of
transcriptional factors ArcA and Fnr in this process. First, we mounted a method for
LPS extraction followed by a hydrolysis step to obtain a highly-purified lipid A that
can be used to study the composition of this molecule by MALDI-TOF. Thus, in
aerobiosis we observed four major signals corresponding to hexa-acylated and
hepta-acylated lipid A molecules and their corresponding hydroxylated species.
Under this environmental condition, hydroxylated species were more abundant
than non-hydroxylated species. In the case of anaerobic cultures we detected the
same four signal, but this relationship is reversed, being more abundant the nonhydroxylated
species. To determine the participation of ArcA and Fnr in the
modulation of covalent modifications of lipid A, we generated ΔarcA and Δfnr
mutant strains and a strain overproducing ArcA (parcA). Our results indicate that
both regulators are involved in controlling structural changes of lipid A in response
to oxygen availability. Additionally, we observed that these regulators control the
incorporation of other covalent modifications into lipid A.
LpxO enzyme (encoded by lpxO gene) is responsible of the hydroxylation of lipid A
in Salmonella. Therefore, we studied the relative expression (anaerobic/aerobic) of
this gene in wild-type S. Enteritidis and their Δfnr and parcA derivatives via
qRT-PCR. Our results indicate that the expression of lpxO depends on oxygen
availability, an observation that is consistent with changes in the levels of lipid A
hydroxylation described herein. Furthermore, ArcA and Fnr participate in the
modulation of the expression of this gene.
The results of this study reveal a change in the hydroxylation degree of S.
Enteritidis lipid A in response to oxygen availability, in which global regulators ArcA
and Fnr are involved. These findings contribute to understanding the molecular
mechanisms of adaptation to the environment used by Salmonella during infection === Fondecyt
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