2. Molecular mechanisms of A. baumannii involved in the
physiology of persister cells
Factors influencing the level of persister cells
Several studies have investigated the formation of persister cells byA. baumannii. The study by Bart et al., (2013) was the first
investigation on this type of cells in A. baumannii . Thirty-seven
clinical strains from the hospital of Porto Alegre (Brazil), isolated
from January to September 2012, were tested for persister cell formation
with two antibiotics, polymyxin B and tobramycin (Table 1) [32].
This study showed the proportion of persister cells strongly depended on
the strain and varied between 0.0007% to 10.1% and 0.0003 % to 11.84
% of the total population, for polymyxin B and tobramycin respectively
[32]. However, for the majority of isolates, the most likely
proportion was about 1.2% for polymyxin B and 0.5% for tobramycin.
In another study about the same hospital [33] twenty other clinical
isolates were classified according to their ability to form biofilms. As
known, the proportion of persister cells in biofilm mode was much higher
than in planktonic mode but surprisingly, strong biofilm producers had
some of the lowest persister levels (Table 1). Persister levels show a
higher heterogeneity (0.2 to 7.2%) for isolates producing low biofilm
compared with those producing more biofilm. Among the isolates, 5 were
tested in planktonic mode at different concentrations (15 to 200 times
the minimum inhibitory concentration (MIC) of meropenem). The proportion
of persisters did not vary as a function of antibiotic concentration.
The authors suggested that this persister level heterogeneity could have
four distinct and non-exclusive origins: the individual ability to
produce persistence stochastically, specific regulatory mechanisms, the
metabolic structure of each isolate and the influence of other previous
stresses.
Persister cells were also observed after ciprofloxacin treatment of non
multidrug resistance (MDR) strong biofilm former clinical isolates
[34]. This treatment also rose the level of ciprofloxacin resistance
in regrown planktonic cells suggesting a link between biofilm,
persistence and resistance (Table 1).
Treatments by aminoglycosides and tobramycin in particular were also
shown to promote persister formation (Table 1) [35], the level of
which was modulated by oxygenation level and composition of the culture
medium [35], [36]. This modulation via nutriment
availability was also observed on persister levels obtained after
ciprofloxacin treatment (Table 1) [36]. The growth phase is another
factor that was shown to strongly influence the proportion of persister
cells [37], but this has not yet been demonstrated for A.
baumannii . Thus, if the level of persister formation depend weakly on
the culture parameters or on the antibiotic used, it was suggested to be
essentially related to the strain in A. baumannii . Indeed, the
diversity of the A. baumannii genomes reflects a high degree of
diversity in the strains themselves (Table 1) [38]. In fact, out of
the 2 500 A. baumannii genomes, only 2 221 genes are shared
between each strain (core genome), out of the 19 272 that correspond to
the pangenome. Strain diversity may be a main explanation for the
difference in persister formation levels.
Toxin/antitoxin systems related to the formation of persister
cells
The toxin/antitoxin (TA) systems are composed of a stable toxin and an
unstable antitoxin, which neutralizes the activity of the toxin. TA
systems are currently grouped into five types (types I to V) according
to the nature of the antitoxin and the mode of interaction between toxin
and antitoxin. In all cases, toxins are proteins, while an antitoxin may
be either RNA or protein [39].
Initially presented as a plasmid maintenance system [40], new
advances have shown that these systems act also on the physiology of the
bacteria. Indeed, they allow slowing down bacterial growth in response
to a stress factor, promoting thus an entry into bacterial
persister state [41]. The involvement of “high persistence”
genes (hipA/hipB TA operon) in the persister phenomenon was
discovered in 1983 in E. coli K12 [42]. This system is the
most documented for its involvement in persister cell formation. Under
unfavorable conditions, a cascade of reactions activates the TA system.
Indeed, the presence of stress induces activation of the stringent
response, leading to 5’-diphosphate 3’-diphosphate (ppGpp) accumulation.
Although not essential, ppGpp accumulation leads to the formation of
polyphosphate (polyP) by the polyphosphate kinase (PPK) [43]. This
polyP then interacts with the protease complex Lon, activating a complex
polyP - Lon that promotes protein degradation of the HipB antitoxin from
the TA. HipA toxin is thus released and inhibits protein synthesisvia an inhibition of the glutamyl-tRNA synthetase [44]. This
leads to an accumulation of uncharged tRNAGlu in the cell and results in
failure of protein biosynthesis. The TA systems remain partially
described in A. baumannii and present, as already described, a
significant variability depending on the strain [45], [46],
[47].
The study of 476 genomes of A. baumannii isolates predicted 15
different TA systems. They were detected either on the strain genome or
on their plasmids (ACICU, AYE, SDF, MDR-ZJ06, p3ABAYE…) [45]. The
authors subcloned the toxins and expressed them in E. coli under
an inducible promoter. They then investigated the toxic effect of the
protein and whether it was functional. In this group, five of them
appear to be functional (Table 1). These five TA systems are all type
II. Three of these TA are orthologs of the bacterial and archaeal
systems RelB/RelE, HicA/HicB and HigB/HigA, and two are unique TA
modules, AbkA/AbkB (also called SplT/SplA) and CheT/CheA [45],
[48], [49].
AbkA/AbkB : Among these five potential TA systems, the
AbkA/AbkB system was the only system studied for its involvement in
persister formation in A. baumannii . It is mainly carried by
plasmid but does not appear to be directly involved in plasmid
maintenance [50], [51], [52]. The toxin AbkB is an
endoribonuclease that inhibits translation, and AbkA as its cognate
antitoxin [50]. The genes abkA/abkB were over-expressed (2.7
fold change) in persister cells under antibiotic treatment (10 × MIC of
imipenem) [53]. The strains overexpressed AbkA and presented a high
proportion of persister while a drastic decrease was observed in the
other strains without this system. It is also interesting to note that a
significant correlation was observed between the presence of abkA and blaOXA-24 genes in carbapenem-resistant isolates obtained
from patients in Iran [48]. In this case, it was suggested a
potential role for these systems as plasmid stabilization systems,
contributing to the evolution of antibiotic resistance in A.
baumannii [45], [49].
Concerning the other TA systems, their function was hypothesized
regarding their identity with already described TA systems in different
bacteria.
HigA/B : Indeed, for HigB/HigA, no link has been yet
established for its involvement in A. baumannii persisters.
However, in Mycobacterium tuberculosis it is involved in stress
response [54]. In Pseudomonas aeruginosa, the toxin HigB
regulates virulence factor production (e.g pyocyanin production,
pyochelin, swarming mobility) and promote biofilm formation where a high
proportion of cells are known to persist [55], [56]. HigA
antitoxin represses the expression of a key transcriptional regulator of
the virulence systems by directly binding to their promoter regions, and
it controls pyocyanin synthesis, type III secretion system (T3SS), and
type VI secretion system (T6SS) expression [56]. In A.
baumannii, the HigB2/HigA2 system was found on the 11kb plasmid pAB120
also carrying genes for resistance to carbapenems [57]. The
hetero-oligomer has a feedback control on its expression. It was also
modulate by environmental stresses and was shown to be overexpressed in
stationary phase or under iron deficiency [57]. Both HigB/HigA and
AbkA/AbkB are the most abundant systems, and only detected to be carried
by plasmids up to now. They are predominantly present in isolates from
the international clones one and two (IC-I and IC-II) [45],
[48], [57], [58], [59].
CheT/CheA : Regarding A. baumannii CheT/CheA
system, CheA presented 25 % identity with the E. coli DinJ
antitoxin, and has 37 % identity with an N-acetyltransferase
superfamily [48]. . The predicted structure of the protein seems to
show that CheA possesses helix-turn-helix (HTH) domains characteristic
of DinJ. In E. coli , DinJ/YafQ system is involved in the
formation of persister cells [60]. Kill-rescue assays on A.
baumannii isolates showed that CheT/CheA served as a TA module: the HTH
domain of the protein CheT acted as a toxin and the GNAT domain of CheA
protein neutralized the HTH effect acting thus as an antitoxin [48].
RelB/RelE : The RelB/RelE system is detected at
genomic level in some A. baumannii clinical strains (AC1633,
ACICU, MDR-ZJ06, TCDC-AB0715, TYTH1 or AB 5075) [38], [45],
[46], [61]. In E. coli , the target of the RelB toxin is
the A ribosomal site. The blockage of this site promotes the cleavage of
the messenger ribonucleic acid (mRNA) during translation [62]. This
allows a translation stop and thus a stop of protein production[62].
HicA/B: The last TA system hicA/hicB was shown
to be overexpressed in A. baumannii ATCC 17978 upon ciprofloxacin
treatment (50 × MIC), suggesting an involvement of this system in the
response of bacteria to antibiotic shocks [63]. In E. coli K12, the toxin HicA cleaves mRNA and also the tRNA in a
ribosome-independent manner and HicA is reported to be inactivated by
HicB [64]. In Burkholderia pseudomallei , this system was
suggested to be involved in the formation of persister cells [65].
Finally, a Zeta/Epsilon system has been predicted and detected in the
plasmids p2ABTCDC, pAbSK-OXA-82, pABTJ1 and pACICU2 [45]. Zeta
toxins are notably larger (360 amino acids) than their currently known
functional orthologs (270 amino acids). The Zeta toxin in A.
baumannii could block cell wall biosynthesis by phosphorylation of the
peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG)
[66]. Indeed, Zeta would form an ATP-UNAG-Zeta complex. This complex
will block the action of MurA, responsible for peptidoglycan
biosynthesis [67], which is the initial step in membrane
biosynthesis [66]. Zeta then appears to have a strong implication in
cell multiplication and would be an interesting target, as it stops
bacteria to grow rapidly. Although not an antitoxin-toxin system, PPK
kinase is also involved in protein degradation, particularly in
antitoxin degradation. A study in A. baumannii investigated the
effect of polyP via a mutant of PPK [68]. In this study, ATCC
17978 strains deficient in PPK1 (Dppk1::Apr) and supplemented with PPK1
(Dppk1::Apr/PJL02-ppk1) were studied. As expected, the authors showed
that PPK1 is essential for polyP formation in vivo . The PPK1
mutant had a reduced motility by a destabilisation of the pili
structure, inhibited biofilm formation and decreased persister under
antibiotic (40 × MIC of Ampicillin) but also under other stresses such
as H2O2, heat shock and starvation
stress. These phenotypes were restored by complementation.
Interestingly, metabolomics analysis revealed that PPK1 was associated
with glycerophospholipid metabolism and fatty acid biosynthesis. The
authors suggested thus alteration of glycerophospholipid metabolism
could alter the membrane charge, resulting in a resistance of bacteria
to the tested stresses [68].