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Slow growing parental strains (SCVs)
Compensated strains
Figure 3. Fitness of compensated mutants and the respective parental
mutant (strain and mutation indicated below the chart) relative to the
wild-type strain. The tness of the wild-type strain was set to 1.
10
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HNP-1 (mg/L)
DA6192
(wild-type)
DA10887
(hemC T241P)
100
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100
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(a)
(b)
DA10887
(hemC T241P)
DA6192
(wild-type)
Figure 2. Killing effect of (a) lactoferricin B (414) and (b) HNP-1 on the
protamine-resistant hemC mutant DA10887 (an SCV) and the susceptible
parental strain DA6192. The curves represent the change in the
percentage of viable bacteria over time relative to that at the start of
the experiment.
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involvement in respiration (e.g. fmt and rplQ mutants) were not
resistant to protamine.
The mutations responsible for protamine resistance were
identied in 22 of the 23 mutants. Nineteen mutants carried
mutations in genes involved in four different steps of haem bio-
synthesis. These mutations were found at 14 different locations
in the genes. The variety of different mutations conrms the
presence of a large mutational target and explains the relatively
high mutation rate. Furthermore, it indicates that it is the lack of
the end product of the pathway, haem, that causes resistance
and not the individual proteins or metabolic intermediates of
the pathway. In bacteria, haem is involved in multiple cellular
functions including respiration and regulation of gene expression.
Haem serves as cofactor for many cytochromes and catalases,
and sirohaem and cobalamin production is dependent on inter-
mediates of the haem biosynthetic pathway.
31,34,35,43
Mutations
in the different hem genes lead to defects in electron transport
and respiration. This in turn causes slow growth by reducing
the amount of ATP available for metabolism and leads to a
decreased transmembrane potential, reducing the afnity for
and uptake of cationic compounds, explaining the observed pro-
tamine resistance.
17
Three of the protamine-resistant mutants carried a nonsense
mutation at the same position of cydC. These mutants had a
growth rate that was 15% lower than that of the wild-type
strain and were among the least resistant mutants. In
Escherichia coli, cydC encodes a subunit of an ATP-binding cas-
sette transporter (CydDC).
32,33
The sequence similarity of the
E. coli and Salmonella Typhimurium LT2 cydC genes is 75% at
the nucleotide level, and the protein probably has a similar func-
tion in the two strains. Goldman et al.
44
found the periplasm of
an E. coli cydC mutant to be more oxidized than that of the wild-
type strain and it was suggested that the CydDC transporter
maintains redox balance by exporting reducing molecules to
the periplasm. It was later demonstrated that cysteine and the
antioxidant glutathione are substrates for the transporter.
32,33
The lowered reducing power in the cydC mutants is thought to
affect several important functions, including assembly of c-type
cytochromes and functioning periplasmic cytochrome b.
33
These defects will in turn affect several cellular functions includ-
ing electron transport, which would explain the lower protamine
susceptibility. The less severe phenotype changes in these
mutants might reect redundancy in the regulation of redox
homeostasis.
The hemA and hemL gene products are involved in production
of 5-ALA, an intermediate in the haem biosynthetic pathway.
35
Protamine resistance and growth defects of the examined
hemL and hemA mutants could be reversed by addition of ALA
to the growth medium, establishing the connection between
defects in ALA production and the resistance phenotype of
these mutants. It has been observed that ALA can be produced
non-enzymatically in a hemL-independent fashion, which may
explain the somewhat higher tness observed for the hemL
mutants compared with other hem mutants.
36,45
An alternative
explanation would be that the defective enzyme retains residual
activity, allowing a low level of ALA production. Furthermore,
hemA is the rst gene in an operon and expression of the down-
stream genes might, due to polarity effects, be altered in the
Table 5. Characterization of compensatory mutations in independently passaged lineages of protamine-resistant mutants
Parental strain, resistance
mutation and amino acid change
Growth-compensating mutation
and amino acid changes Generations of growth MIC (mg/L)
DA6192 (wild-type) NA not passaged 95
DA10886 hemL (ccc!tcc, P297S) 140 95
hemL (acc!ccc, T297P) hemL (ccc!tcc, P297S) 150 ND
hemL (ccc!tcc, P297S) 170 ND
hemL (ccc!tcc, P297S) 360 ND
no compensation, 6 lineages 370 NA
DA10887 hemC (ccc!aac, P241N) 21 ND
hemC (acc!ccc, T241P) extragenic 14 375
hemC (ccc!cac, P241H) 41 95
hemC (possible amplication) 28 95
hemC (possible amplication) 56 62.5
hemC (ccc!ctc, P241L) 7 ND
not identied, 5 lineages 21/21/35/63/70 ND
DA10888 reversion 14 95
hemA (gtc!ggc, V45G) reversion 84 ND
reversion 224 ND
not identied 30 95
no compensation, 6 lineages 260 NA
DA10891 del prpE-hemB-yaiU no compensation 260 NA
ND, not determined; NA, not applicable.
Protamine resistance in Salmonella Typhimurium LT2
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hemA mutants, resulting in tness costs unrelated to the pro-
duction of ALA.
As expected, ALA supplementation had no effect on hemC
and hemG mutants impaired in haem biosynthetic reactions sub-
sequent to completion of ALA synthesis. In addition, a mutant
defective in the rst steps of sirohaem and cobalamin synthesis
was not resistant to protamine in either the presence or absence
of ALA, enabling us to rule out involvement of these two pro-
ducts in protamine resistance. Protamine susceptibility of a
haemin-permeable hemA mutant could be reversed by exogen-
ous haemin, further indicating that haem deciency caused
the resistance phenotype.
Mutants decient in respiratory genes have been isolated
from clinical specimens from various sites of the body and are
being increasingly recognized as a major contributor to persist-
ent and recurrent diseases such as osteomyelitis, cystic brosis
and foreign body-related infections.
17,19
These SCVs have an
improved capacity to survive within cells and are more resistant
to many antimicrobials, including aminoglycosides.
20,38,46,47
Similar to clinical SCVs, all examined protamine-resistant
mutants were less susceptible to aminoglycosides than the par-
ental strain, irrespective of whether they carried hem or cydC
mutations. Aminoglycoside transport over the bacterial mem-
brane is dependent on a charge differential across the mem-
brane, formed during electron transport and respiration.
19,39
Thus, the respiratory defects of the mutants interrupt uptake
and function of the antibiotic. The mutants were also less sus-
ceptible to the lipopeptide antibiotic colistin. Colistin activity is
dependent on electrostatic interactions between the antibiotic
and the negatively charged bacterial membrane, and, following
the same reasoning as above, the respiratory defects of the
mutants probably produce changes in the bacteria that reduce
this interaction.
48
It has been previously observed that SCVs of
S. aureus with different auxotrophisms are resistant to the AMP
lactoferricin B.
49
Similarly, we found a Salmonella Typhimurium
LT2 SCV (hemC T241P) to be less susceptible to this peptide.
Samuelsen et al.
49
suggested that lactoferricin resistance was
caused by a reduced metabolic activity rather than by decreased
membrane interaction and uptake, based on the observation
that labelled peptide was efciently internalized by the SCV.
The examined hemC mutant was also less susceptible to the
human defensin HNP-1. Together with HNP-2 and 3, this
peptide accounts for 5% of the human neutrophil proteins.
2,30
Other immune cells, e.g. T cells, also express HNP-1, and it seems
likely that resistance could facilitate bacterial survival in the
human body.
2
That is, respiratory defects of SCVs can increase
resistance to several antimicrobial agents, including peptides
present in the human body, and probably does this through
several different mechanisms.
Clinically isolated SCVs are often described as unstable, fre-
quently reverting to a normal colony phenotype. Generally, pro-
tamine resistance conferred a high tness cost displayed as a
reduced growth rate. The majority of the mutants appeared to
be phenotypically stable, but occasionally certain mutants
re-established fast growth. For some mutant strains it was poss-
ible to select variants with increased tness and second-site
mutations by serial passage of the mutants in the absence of
selection. For the examined hemA and hemL mutants only a
few of the individual lineages compensated and a substantial
fraction of these were genetic revertants. No compensation
was observed for a strain with a deletion of hemB, whereas, in
contrast, all lineages of strain DA10887 (hemC, T241P) compen-
sated rapidly. The growth rates of these lineages were signi-
cantly lower than that of the wild-type strain, indicating that
they did not revert back to the parental genotype. When the
hemC gene of these mutants was sequenced, some strains
were found to have a double DNA sequence at specic positions
in the gene, indicating the presence of a duplication and sub-
sequent mutational divergence. This in turn provides a possible
explanation for the fast compensation of all DA10887 lineages.
The hemC gene is located between two rRNA operons in an
area of the chromosome known to undergo frequent dupli-
cations (sequence repeats provide substrate for duplications),
which may play a role in the fast compensation in these
strains.
50
If so, this would provide a novel mechanism for the
reported phenotypic instability of many SCVs. The potential
occurrence of gene duplications in these strains is currently
under investigation.
In the present study, all isolated protamine-resistant mutants
have the typical characteristics of SCVs. They grow slowly and
carry resistance mutations in genes associated with redox reac-
tions and respiration, are resistant to aminoglycosides, other
antibiotics and peptides, and some are also genetically unstable.
A large tness cost, as observed for the mutants in this study,
would normally imply a competitive disadvantage in the
absence of selective pressure, and it is expected that such
strains would be outcompeted by faster growing strains.
However, SCVs are frequently recovered from patients and have
been suggested to be an important part of the disease pro-
gression and survival in the host for intracellular pathogens.
Thus, the low tness observed might actually provide a survival
advantage in this case. Spontaneously formed SCVs of
Salmonella have only been isolated and characterized in a few
studies, but, as emphasized by Proctor and co-workers in their
studies of SCVs in S. aureus, certain AMPs in the body might
select for or stabilize the SCV phenotype.
17,18,20
Formation of
SCVs could be a way for the bacteria to avoid killing by certain
AMPs, and could be a potential problem for the use of pharma-
ceuticals based on AMP structures whose mode of action is
based on an energized bacterial membrane or actively growing
bacteria.
Acknowledgements
We thank Diarmaid Hughes and Annika Nilsson for comments.
Funding
This work was supported by grants from the Swedish Research Council
(2009-3486), 6th EU Framework Program (2005-318152) and Vinnova
(2009-00169) to D. I. A.
Transparency declarations
None to declare.
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Supplementary data
Table S1, Figure S1 and Figure S2 are available as Supplementary data at
JAC Online (http://jac.oxfordjournals.org/).
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