The Moraxella catarrhalis AdhC–FghA system is important for formaldehyde detoxification and protection against pulmonary clearance

Abstract

Multidrug-resistant clinical isolates of Moraxella catarrhalis have emerged, increasing the demand for the identification of new treatment and prevention strategies. A thorough understanding of how M. catarrhalis can establish an infection and respond to different stressors encountered in the host is crucial for new drug-target identification. Formaldehyde is a highly cytotoxic compound that can be produced endogenously as a by-product of metabolism and exogenously from environmental sources. Pathways responsible for formaldehyde detoxification are thus essential and are found in all domains of life. The current work investigated the role of the system consisting of the S-hydroxymethyl alcohol dehydrogenase (AdhC), a Zn-dependent class III alcohol dehydrogenase, and the S-formyl glutathione hydrolase (FghA) in the formaldehyde detoxification process in M. catarrhalis. Bioinformatics showed that the components of the system are conserved across the species and are highly similar to those of Streptococcus pneumoniae, which share the same biological niche. Isogenic mutants were constructed to study the function of the system in M. catarrhalis. A single fghA knockout mutant did not confer sensitivity to formaldehyde, while the adhC–fghA double mutant is formaldehyde-sensitive. In addition, both mutants were significantly cleared in a murine pulmonary model of infection as compared to the wild type, demonstrating the system’s importance for this pathogen’s virulence. The respective phenotypes were reversed upon the genetic complementation of the mutants. To date, this is the first study investigating the role of the AdhC–FghA system in formaldehyde detoxification and pathogenesis of M. catarrhalis.

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Vol.:(0123456789)
Medical Microbiology and Immunology (2024) 213:3
https://doi.org/10.1007/s00430-024-00785-0
ORIGINAL INVESTIGATION
The Moraxella catarrhalis AdhC–FghA system isimportant
forformaldehyde detoxification andprotection againstpulmonary
clearance
DinaOthman1· NohaM.Elhosseiny2· WafaaN.Eltayeb3· AhmedS.Attia2
Received: 21 October 2023 / Accepted: 24 January 2024
© The Author(s) 2024
Abstract
Multidrug-resistant clinical isolates of Moraxella catarrhalis have emerged, increasing the demand for the identification
of new treatment and prevention strategies. A thorough understanding of how M. catarrhalis can establish an infection and
respond to different stressors encountered in the host is crucial for new drug-target identification. Formaldehyde is a highly
cytotoxic compound that can be produced endogenously as a by-product of metabolism and exogenously from environmental
sources. Pathways responsible for formaldehyde detoxification are thus essential and are found in all domains of life. The
current work investigated the role of the system consisting of the S-hydroxymethyl alcohol dehydrogenase (AdhC), a Zn-
dependent class III alcohol dehydrogenase, and the S-formyl glutathione hydrolase (FghA) in the formaldehyde detoxifica-
tion process in M. catarrhalis. Bioinformatics showed that the components of the system are conserved across the species
and are highly similar to those of Streptococcus pneumoniae, which share the same biological niche. Isogenic mutants were
constructed to study the function of the system in M. catarrhalis. A single fghA knockout mutant did not confer sensitivity to
formaldehyde, while the adhC–fghA double mutant is formaldehyde-sensitive. In addition, both mutants were significantly
cleared in a murine pulmonary model of infection as compared to the wild type, demonstrating the system’s importance for
this pathogen’s virulence. The respective phenotypes were reversed upon the genetic complementation of the mutants. To
date, this is the first study investigating the role of the AdhC–FghA system in formaldehyde detoxification and pathogenesis
of M. catarrhalis.
Keywords Moraxella catarrhalis· Formaldehyde resistance· S-hydroxymethyl alcohol dehydrogenase· S-formyl
glutathione hydrolase· Pulmonary clearance
Introduction
Moraxella catarrhalis, previously considered a commensal
microorganism, has been commonly implicated as the major
etiological agent of otitis media and sinusitis in children,
and in exacerbation of chronic obstructive pulmonary dis-
ease in adults [1]. Multidrug-resistant clinical isolates of
M. catarrhalis have emerged, increasing the demand for
the identification of new treatment and prevention strate-
gies against this pathogen, especially in the absence of an
efficient vaccine [2, 3].
A formaldehyde detoxification system is essential for
microorganisms to protect themselves from cytotoxic formal-
dehyde [4]. In the human body, formaldehyde is produced
during the metabolism of methanol, adrenaline, creatine, and
histones. Most importantly, it is a by-product of some chemi-
cal reactions associated with the immune response, such as
Edited by: Isabelle Bekeredjian-Ding.
* Ahmed S. Attia
ahmed.attia@pharma.cu.edu.eg
1 Graduate Program, Department ofMicrobiology
andImmunology, Faculty ofPharmacy, Cairo University,
Cairo11562, Egypt
2 Department ofMicrobiology andImmunology, Faculty
ofPharmacy, Cairo University, Room #D404, Kasr El-Ainy
Street, Cairo11562, Egypt
3 Department ofMicrobiology, Faculty ofPharmacy, Misr
International University, Cairo19648, Egypt
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Medical Microbiology and Immunology (2024) 213:3 3 Page 2 of 10
the methylation of histamine. During the respiratory burst by
macrophages and neutrophils to kill pathogens, superoxide and
hydrogen peroxide are generated, which in turn react with bac-
terial iron-sulfur clusters to produce free radicals. These free
radicals react with sugars to produce formaldehyde as a toxic
end product that could be used to fight invading pathogens [4].
Formaldehyde is also produced as a part of bacterial metabo-
lism in cell biological processes [5]. For all of the aforemen-
tioned reasons, the formaldehyde detoxification processes are
required to avoid formaldehyde lethal and mutagenic effect [6].
Formaldehyde detoxification is achieved through three
mechanisms; thiol-dependent, ribulose monophosphate-
dependent, and pterin-dependent mechanisms [4]. The glu-
tathione-dependent repair system, also known as the thiol-
dependent pathway, appears to be widely spread in nature
and has been found in most prokaryotes, and all eukaryotes
[7]. In the majority of microorganisms, the thiol is the trip-
eptide glutathione. Initially, glutathione binds to formalde-
hyde to formS-hydroxymethylglutathione [8]. This reaction
occurs spontaneously in most microorganisms, with some
exceptions where this reaction is catalyzed by a glutathione-
dependent formaldehyde-activatingenzyme, Gfa [9, 10].
The S-hydroxymethylglutathione adduct is then oxidized
by a zinc-containing, nicotinamide adenine dinucleotide
(NAD+)-dependent alcohol dehydrogenase, AdhC, to gener-
ate the thioesterS-formylglutathione [11]. Finally, formate is
produced and glutathione is regenerated upon the hydrolysis of
S-formyl glutathione. This last step is catalyzed by an esterase,
EstD, orS-formylglutathione hydrolase, FghA [12].
Numerous studies have been investigating the genetic fac-
tors involved in formaldehyde detoxification and their role in
stress protection, bacterial virulence, and biofilm formation
in different microorganisms [6, 7, 13–19]. For example, the
glutathione-dependent formaldehyde detoxification system
AdhC–EstD was shown to be important for the optimum via-
bility of Neisseria meningitidis in biofilm communities [20]. In
another study, the loss of the encoding gene of EstD in N. gon-
orrhoeae caused an impairment in the ability of this organism
to survive within human cervical epithelial cells [17]. While
many of the genetic factors contributing to the physiology and
virulence of M. catarrhalis have been identified [21–23], the
role of formaldehyde detoxification in these processes has not
been yet investigated. In this study, the adhC and fghA genes
of M. catarrhalis have been investigated with respect to their
potential role in the formaldehyde detoxification and the viru-
lence of this pathogen.
Materials andmethods
Ethics statement
Animal procedures were approved by the Research Ethics
Committee of the Faculty of Pharmacy, Cairo University,
Approval No. MI (2510), following the Guide for the Care
and Use of Laboratory Animals published by the Institute
of Laboratory Animal Research, USA.
Bacterial strains andculture conditions
M. catarrhalis O35E was kindly provided by Dr. Eric
J. Hansen [24] and it was used as the wild type (WT).
The derivatives were all generated in its background. M.
catarrhalis strains were grown on Tryptic Soy Agar (TSA)
or Columbia blood agar at 37°C and 5% CO2 in a carbon
dioxide incubator (Binder, Germany), or in Tryptic Soy
Broth (TSB) at 37°C with shaking at 180rpm under aerobic
conditions [25]. Escherichia coli DH5-α, used as a cloning
host, was grown at 37°C in TSB with shaking at 180rpm,
or on TSA. When needed, media were supplemented with
kanamycin at a final concentration of 15µg/mL, streptomy-
cin at a final concentration of 250µg/mL, and ampicillin at
a final concentration of 100μg/mL.
Bioinformatics analyses
The protein sequenceof the EstD of N. meningitidis (NCBI
protein id CAM08666.1) served as a template for a BlastP
analysis [26], to identify homologs of this esterase inM.
catarrhalis O35E.To survey the conservation of the pro-
tein, the BlastP search was also extended to all the strains
available in M. catarrhalis taxid 480. Sequences of pre-
viously studied FghA homologs were retrieved from the
NCBI (TableS1), and the same was performed with the
AdhC homologs (TableS2). Then, multiple sequences’
alignment with the Blast-retrieved FghA of M. catarrhalis
O35E (NCBI Protein id EGE27440.1) as a query sequence
was carried on using Clustal Omega [27] applyling the
default parameters. Phylogenetic trees were constructed via
NGphylogeny.fr web tool, which employs multiple align-
ment using fast Fourier transform (MAFFT) for multiple
sequence alignment, Block Mapping and Gathering with
Entropy (BMGE) for alignment curation, Phylogeny soft-
ware for the maximum-likelihood principle (PhyML) for
tree inference, and finally Newick display for tree render-
ing [28–32]. To calculate the percent similarity and identity,
EMBOSS Needle tool was used with the default parameters
[33]. To further confirm the interaction between AdhC and
FghA and show possible interactions with other proteins, the
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Medical Microbiology and Immunology (2024) 213:3 Page 3 of 10 3
protein–protein functional interaction analysis tool STRING
[34] was used. To investigate if the adhC and fghA genes
form an operonic pair as consistently reported for the sys-
tem, the operon prediction tool Operon Mapper [35] was
used applying the default parameters, and the whole M.
catarrhalis O35E genome (AERL00000000.1) as a query
sequence.
Construction ofM. catarrhalis ΔfghAand ΔadhC–
fghA deletion mutants andrescue strains
Using theM. catarrhalisO35E chromosomal DNA as a tem-
plate, the primer pairs DO001–DO002 and DO003–DO004
(TableS3 and Fig.1) were used to amplify a 1002 and a 1006
base pair fragments upstream and downstream of thefghA
open-reading frame (ORF), respectively. The fragments were
then digested using XmaIand ligated together. The ligation
product was used as a template for a second PCR reaction
using primer pair DO001–DO004. The product was then
ligated into the rapid cloning vector pJET1.2/blunt (Thermo
Fisher Scientific, Lithuania), to yield plasmid pJET-U + D.
A non-polar kanamycin resistance cassette was obtained by
digesting plasmid pUC18K [36] with XmaI, followed by
gel purification. The product was then ligated with plasmid
pJET-U + D digested with the same restriction enzyme, then
transformed into E. coli DH5-α and plated on TSA contain-
ing ampicillin and kanamycin. The resultant plasmid was
designated pJET-UkanD. This plasmid was used to amplify
a ~ 3kb construct consisting of the kanamycin cassette
flanked by the fghA upstream and downstream fragments,
and the product was transformed intoM. catarrhalisO35E
as previously described [37]. The homologous recombina-
tion of the mutant construct into the chromosome and the
replacement of the WT fghA gene to yield ΔfghAmutant
was confirmed by a series of PCR reactions using primers
within and outside the mutant construct. To construct the
ΔadhC-fghA double mutant,a similar approach was adopted
but using primer pairs DO008–DO009 and DO003–DO004
Fig. 1 Genetic organization and functional relation between the M.
catarrhalis adhC and fghA. A A schematic diagram showing the
organization of the neighboring ORFs in the genetic loci of the adhC
and fghA in the M. catarrhalis WT O35E genome. The binding posi-
tion of the primers used in this study and the loci tags are indicated.
The direction of the ORF arrows indicates the direction of transcrip-
tion. The ruler above the arrows indicates the size of DNA fragment
in base pairs; bp. The map was generated by Ankh diagram v1.1tool
by HITS Solutions Co. (Bioinformatics Department, Cairo, Egypt).
B A schematic diagram representing the interaction between AdhC
(encoded by EA1_02212) with FghA and other M. catarrhalis pro-
teins including Gdsl-like lipase (encoded by EA1_06621), MsrAB,
and GdhA. The figure was generated using STRING database and the
lines drawn between the functional pairs represent the predicted func-
tional relationship (neighborhood; green, gene fusion; red, co-occur-
rence; dark purple, co-expression; black, databases; teal, text mining;
yellow, experimentally determined interactions; pink line, and protein
homology; light blue)
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Medical Microbiology and Immunology (2024) 213:3 3 Page 4 of 10
to amplify a 329 and a 1002 base pair fragments upstream
and downstream of adhC and fghA, respectively. To repair
the ΔfghAmutant, the fragment amplified with primer pair
DO001–DO004 from the WT O35E M. catarrhalis strain
together with the mutatedrpsLamplicon [38] were trans-
formed into theΔfghAin a congression experiment as pre-
viously described [39]. Isolated colonies that could grow
on streptomycin, and failed to grow on kanamycin, were
selected as potential complemented mutants, confirmed
using PCR, and designated ΔfghA/R. To repair ΔadhC–fghA
mutant, a similar approach was used, but using primer pair
DO008–DO004 to amplify the WTamplicon. The con-
firmed rescue mutant was designated ΔadhC–fghA/R. The
sequences of all the oligonucleotides used in this study are
listed in TableS3.
Growth curve analysis
Colonies of the WT O35E, ΔfghA, ΔfghA/R, ΔadhC-fghA,
and ΔadhC-fghA/Rgrown overnight on Columbia blood
agar were suspended in TSB to an optical density at 600nm
(OD600) ~ 1.0 then diluted 1:50 in 15mL TSB broth. The
cultures were incubated in a shaking incubator at 37°C and
180rpm and the OD600was measured each hour for 8h
using a visible spectrophotometer Jenway 6300 (Jenway,
United Kingdom). Growth curves were constructed by plot-
ting OD600 versus time.
Formaldehyde sensitivity assay
Formaldehyde susceptibility was assessed using the disc
diffusion susceptibility assay as previously described [20].
Briefly, cells of the WT O35E, ΔfghA, ΔfghA/R, ΔadhC-
fghA, and ΔadhC-fghA/Rfreshly streaked on Columbia
blood agar were suspended in TSB to an OD600 ~ 0.4. The
adjusted cell suspensions were spread over TSA plates
(supplemented with kanamycin for ΔfghA and ΔadhC-fghA
mutants, streptomycin for ΔfghA/R and ΔadhC-fghA/Rcom-
plemented mutants) using sterile cotton swabs. Sterile single
Whatmann no. 1 filter paper discs were saturated with 5 μL
of a 5% formaldehyde solution (Piochem, Egypt) and placed
onto the agar surface. The diameter of the inhibition zone
around the disc was measured after an overnight incubation
at 37°C in a CO2 incubator with the petri-dishes inverted.
Formaldehyde sensitivity was also tested by another assay
[20]. Briefly, a bacterial suspension was prepared as in the
sensitivity assay detailed above, and seven tenfold serial
dilutions were prepared in a 96-well microplate in TSB. Five
microliters of each dilution were spotted on TSA plates, sup-
plemented with 0-, 0.8-, or 1-mM formaldehyde. The spots
were left to dry then survival was determined by counting
the number of visible colonies after an overnight incubation
at 37°C in a CO2 incubator with the petri-dishes inverted.
Determination oftheminimum inhibitory
concentration (MIC)
To obtain a more quantitative assessment of the susceptibility
of the five strains under investigation to formaldehyde, we per-
formed an MIC experiment using the standard microdilution
method [40]. Briefly, a 0.5 McFarland standard suspension
of each of the five strains was prepared using freshly grown
bacterial cells, and then, it was diluted 1:10 and 10 µL were
used to inoculate 12 wells containing 190 µL of TSB contain-
ing twofold dilutions of formaldehyde from 1mM.to 0.5µM.
The wells incubated for 24h at 37°C then inspected for
visual growth. The formaldehyde concentration in the first
clear well was considered the corresponding MIC value for
the respective strain.
Protein profiling using sodium dodecyl
sulfate‑polyacrylamide gel electrophoresis
(SDS‑PAGE)
Cells of the five tested strains were grown overnight in TSB
in the presence and absence of 1mM formaldehyde in a shak-
ing incubator, harvested by centrifugation, and resuspended
in sterile saline to an OD600 ~ 1. The cell suspensions were
mixed with a 3 × reducing Laemmli buffer [41]. These sam-
ples were then heated at 95°C for 10min in a thermal cycler
(Boeco, Germany) before loading on a 10% SDS-PAGE gel.
Gels were afterwards stained with Coomassie blue [41], visu-
alized, and photographed using a gel documentation system
(UVP, Germany).
Murine pulmonary clearance model
The pulmonary clearance model in mice was carried out
as previously described [42]. Briefly, five groups (n = 6) of
6–8-week-old female BALB/C mice were infected intranasally
by injecting 40μl of a bacterial suspension (∼5 × 106CFU)
of each of the WT O35E, ΔfghA, ΔfghA/R, ΔadhC-fghA, and
ΔadhC-fghA/R into the nostrils under anesthesia using 250
µL of 25µg/mL 2,2,2-tribromoethanol. Four-and-a-half-hour
post-inoculation, mice were sacrificed by an overdose of the
anesthesia (750 µL of 25µg/mL 2,2,2-tribromoethanol), fol-
lowed by cervical dislocation. The lungs were excised, homog-
enized, serially diluted, and plated on TSB agar. Plates were
incubated for 48h followed by colony counting.
Results
M. catarrhalis possesses anEstD homolog
Using the N. meningitidis EstD as a query, Blastp against
the M. catarrhalis O35E proteome returned a 268 amino
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Medical Microbiology and Immunology (2024) 213:3 Page 5 of 10 3
acid protein (EGE27440.1) annotated as FghA (for S-for-
mylglutathione hydrolase A) as the closest match, with a
query coverage of 96%, and an identity of 54% (Fig.S1).
Results of the NCBI BlastP between FghA of M. catarrhalis
strain O35E (EGE27440.1) and other M. catarrhalis strains
(taxid 480) shows a high degree of conservation with a per-
cent identity range of 91.79–100% (Fig. S2). Alignment of
homologs from different microbial genera, and even higher
eukaryotes showed that all had high identity (40–65%) and
similarity (55–77.7%) with the FghA of M. catarrhalis (Fig.
S3 and TableS1). The highest similarity obtained was with
S. pneumoniae (77.7%). Interestingly, although phylogenetic
analysis showed that the S. pneumoniae FghA is still the
closest evolutionary relative to the M. catarrhalis protein,
those from closely related genera which scored the high-
est similarity such as Neisseria, and Haemophilus clustered
together in a more distant branch of the tree. Meanwhile, the
FghA homolog from Paraccoccus showed a closer relation-
ship to the M. catarrhalis FghA, although it scored less on
sequence similarity than the aforementioned species (Fig.
S4).
We noticed that the ORF upstream of the M. catarrha-
lis fghA (Fig.1A) was annotated as adhC, a gene encod-
ing for S-hydroxymethyl alcohol dehydrogenase, and which
usually forms an operon with fghA as previously reported
[14, 20, 43]. To investigate the relationship between the
two ORFs, first, the operon prediction tool Operon Map-
per was used, and the results obtained demonstrated that
adhC and fghA are expected to form an operon by a high
probability of 0.97. Next, the protein interaction network
database, STRING, was used to investigate if a predicted
functional link between the two proteins is likely. The gen-
erated interaction network indicated that the M. catarrhalis
AdhC and FghA are strongly predicted as functional partners
by a score of 0.999. The two genes, Adhc and FghA, interact
by gene neighborhood, gene fusion, gene co-occurrence, co-
expression, and protein homology (Fig.1B). The network
also revealed the interaction by gene fusion between FghA
with EA1_06621, a Gdsl-like lipase/ccyl hydrolase family
protein. Another interaction by gene neighborhood between
FghA and MsrAB (EA1_03390), a trifunctional thioredoxin/
methionine sulfoxide reductase a/b protein was shown by the
network. The final interaction by gene neighborhood and
co-expression was predicted with GdhA (EA1_02207) an
NADP-specific glutamate dehydrogenase and FghA. It is
worth mentioning that while the confidence score of these
former interactions was relatively low (0.4), the GdhA was
the only protein predicted to interact with both FghA, and
AdhC. Although it lies upstream of adhC, it was not pre-
dicted to form an operon with the pair.
A similar blast analysis to that conducted using the
FghA was done using the M. catarrhalis O35E AdhC pro-
tein sequence. There was a high identity (41–81.7%) and
similarity (49.2%-90.9%) between AdhC of M. catarrhalis
and its homologs in other species (Fig. S5 and TableS2).
S. pneumoniae AdhC also showed the highest similarity
(90.9%) with that of M. catarrhalis, which is also in agree-
ment with the phylogenetic relationship (Fig. S6). As seen
with FghA, the AdhC proteins of closely related species
diverged from that of M. catarrhalis.
The putative adhC–fghA operon contributes
toformaldehyde detoxification inM. catarrhalis
To investigate the possible role of adhC and fghA in formal-
dehyde detoxification in M. catarrhalis, deletion mutants
lacking the fghA gene (ΔfghA) and both genes fghA and
adhC (ΔadhC-fghA) were constructed, and along with their
complemented mutants (ΔfghA/R and ΔadhC-fghA/R) were
tested for their formaldehyde sensitivity compared to WT
O35E using a disc diffusion susceptibility assay. There was
no significant difference between the five strains in sensitiv-
ity to formaldehyde using this susceptibility assay (Fig.2).
A more sensitive susceptibility assay was then performed
by plating serial dilutions of the five strains on a solid
medium containing increasing concentrations of formalde-
hyde (0, 0.8, and 1mM). Especially at the 1mM formalde-
hyde concentration, the growth of the ΔadhC-fghA was sig-
nificantly attenuated (Fig.3), while this growth defect was
not observed with the rescue strain ΔadhC-fghA/R which
reverted to the WT phenotype. Conversely, the single mutant
ΔfghA and its complemented mutant ΔfghA/R did not dis-
play significant sensitivity to formaldehyde at any of the
tested concentrations. It is worth mentioning here that all
constructed mutants showed no growth defects in compari-
son to O35E when the growth was monitored logarithmically
in plain TSB (Fig.S7).
Upon determination of the formaldehyde MIC for the five
strains, results very similar to those observed above were
obtained. For each of the four strains WT O35E, ΔfghA,
ΔfghA/R, and ΔadhC-fghA/R, the MIC value was 1mM.
On the other hand, ΔadhC-fghA exhibited much lower MIC
value of 7.8µM.
Loss oftheformaldehyde detoxification system
does notalter theprotein expression profile inM.
catarrhalis onSDS‑PAGE
To investigate whether the loss of AdhC and FghA could
alter the expression profile of some M. catarrhalis proteins,
especially given the results of the predicted interactions
obtained in silico from STRING which indicated the pres-
ence of a possible co-expression relationship between some
of the protein pairs, the profiles following incubation with
formaldehyde were visually assessed using SDS-PAGE. As
the only co-expression interactions noticed in silico were
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Medical Microbiology and Immunology (2024) 213:3 3 Page 6 of 10
between FghA with AdhC and FghA with Gdha, no promi-
nent differences could be visually detected between the
WT and any of the ΔadhC-fghA and the ΔfghA mutants or
their complemented counterparts whether with or without
formaldehyde (Fig.S8). This indicates that any differences
that might exist are much more subtle to be detected using
Coomassie blue staining of SDS-PAGE gels.
The AdhC–FghA system isessential forpulmonary
colonization byM. catarrhalis
To investigate whether the role of the AdhC–FghA system
in formaldehyde detoxification would prove important to
the fitness of M. catarrhalis in pulmonary infection, an M.
catarrhalis pulmonary clearance model was conducted.
Interestingly, mice were significantly more capable of clear-
ing both ΔfghA and ΔadhC-fghA to a higher extent than WT
O35E and the complemented mutants with an average one-
log cycle change in the obtained colony counts (Fig.4).
These invivo results indicate that AdhC–FghA system sig-
nificantly contributes to the pathogenesis of M. catarrhalis.
Discussion
Formaldehyde is highly cytotoxic to living organisms, so
they need systems to detoxify formaldehyde to be able to
survive. Several researchers investigated the formaldehyde
detoxification mechanisms and the proteins involved to cope
with this stress [4, 6, 7, 16, 18, 20]. However, the current
study investigates the role of AdhC and FghA proteins in
formaldehyde detoxification in M. catarrhalis.
M. catarrhalis,previously known as N. catarrhalis,
resembles commensalNeisseriaspp. in culture, phenotype,
and ecological niche [44]. Therefore, Neisseria spp. previ-
ously characterized esterase protein EstD seemed to be a
good template to search for similar formaldehyde detoxi-
fying protein in M. catarrhalis. Our results indicated the
high conservation of AdhC and FghA proteins across M.
catarrhalis strains in addition to the species surveyed in
silico in this study. These results could be attributed to the
importance of the system for stress tolerance [4, 7, 20].
This was especially true for S. pneumoniae, which shared
the highest similarity and evolutionary relationship with the
M. catarrhalis proteins. The striking resemblance between
Fig. 2 Loss of the adhC–fghA
operon or fghA gene did not
impair formaldehyde detoxifi-
cation in M. catarrhalis when
tested by disc diffusion assay.
A Photographs of the zones
of inhibition were taken using
a gel documentation system
(UVP) with a ruler under each
strain that represents the scale
in cm. B Box plot representing
results of the formaldehyde disc
diffusion assay, plotting the
zone diameter obtained in cm.
The data represent the mean of
three independent experiments,
and the bars span the difference
between the minimum and max-
imum readings. The line inside
the box represents the median.
The graph was generated using
GraphPad prism version 9.0.0
(GraphPad Software, San
Diego, CA, USA). Statistical
analysis of the data was done
applying one-way ANOVA fol-
lowed by Tukey’s multiple com-
parison test. The “ns” stands for
“non-significant”
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Medical Microbiology and Immunology (2024) 213:3 Page 7 of 10 3
these two organisms, which are considered two major causes
of acute otitis media, could be due to their shared habitat.
They share the same environment within the host, and this
could have driven the development of comparable protective
strategies to combat the same stresses encountered in the
nasopharynx and upper respiratory tract [44].
The interaction network of AdhC and FghA with each
other showed, most importantly, gene neighborhood, co-
expression, and protein homology. This gave plausible evi-
dence supporting the potential roles assigned to these pro-
teins, and their functional relationship. The network also
revealed the interaction by gene fusion between FghA with
other proteins like that encoded by EA1_06621, a Gdsl-like
lipase/ccyl hydrolase family protein. In addition, the MsrAB
(EA1_03390), a trifunctional thioredoxin/methionine sul-
foxide reductase a/b protein that has an important role as
a repair enzyme for proteins that have been inactivated by
oxidation [45] was predicted to have a gene neighborhood
interaction with the FghA. The MsrAB mode of function is
closely related to the mechanism of formaldehyde detoxifi-
cation through the redox potential of glutathione. Another
notable interaction was predicted with GdhA (EA1_02207),
a NADP-specific glutamate dehydrogenase, that belongs to
the Glu/Leu/Phe/Val dehydrogenases family. Glutamate
metabolism plays an essential role in the synthesis of glu-
tathione and Gdh-null mutants generally show a higher
sensitivity to oxidative stress as well as a more rapid deple-
tion of glutathione. These functions support the involvement
of GdhA in the thiol-dependent pathway of formaldehyde
detoxification [46, 47].
As expected, the Operon Mapper data indicated that adhC
and fghA very likely constitute an operon. These findings
are consistent with previous reports that also point out that
these two genes form an operon in other species [4, 7, 14,
20, 43]. As per previous bioinformatic analysis, an attempt
to confirm the function of adhC and fghA in formaldehyde
detoxification in M. catarrhalis was designed. At first, a
single mutant ΔfghA was constructed. There was no signifi-
cant difference between the wild-type and the single mutant
ΔfghA in the formaldehyde sensitivity tests, so a double
mutant ΔadhC-fghA was constructed. A study conducted by
Chen and co-workers mentioned that their single mutant of
the fghA homolog was more sensitive to formaldehyde than
their double mutant [20]. On the contrary, the current study
shows that the increase in sensitivity is more significant
in the double mutant ΔadhC-fghA than the single mutant
ΔfghA. The high sensitivity to formaldehyde in the double
mutant was expected as both proteins interact together in
the formaldehyde detoxification pathway, while in the single
mutant, the results showed that FghA is not essential on its
own to detoxify formaldehyde. One speculation could be
that, in the adhC–fghA mutant, due to the inactivation of
Fig. 3 Loss of the adhC–fghA
operon but not fghA impairs
formaldehyde detoxification
in M. catarrhalis when tested
by serial dilution susceptibility
assay. A Photographs of dilu-
tions on TSA plates containing
increasing concentrations of for-
maldehyde (0mM, 0.8mM, and
1mM) were taken using a gel
documentation system (UVP).
B Box plot graphs of bacterial
counts in CFU/mL with the
different formaldehyde con-
centrations. The data represent
the mean of three independent
experiments, and the bars span
the difference between the mini-
mum and maximum readings.
The line inside the box repre-
sents the median. The graphs
were generated using GraphPad
prism version 9.0.0 (GraphPad
Software, San Diego, California
USA). Statistical analysis of the
data was done applying one-way
ANOVA followed by Tukey’s
multiple comparison test
(*P ≤ 0.05), (**P ≤ 0.01). The
* indicates that the difference is
statistically significant
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Medical Microbiology and Immunology (2024) 213:3 3 Page 8 of 10
theadhC, the accumulation of S-hydroxymethyl glutathione,
the substrate of AdhC, is more toxic to the cells than the
accumulation of S-formyl glutathione, the substrate of FghA,
in the fghA mutant [4]. A complementary explanation for
these results which would account for the non-toxicity of
S-formyl glutathione could be attributed to the presence of
another enzyme that participates with FghA, or completely
replaces it, in the hydrolysis of S-formylglutathione to glu-
tathione and formate. This explanation was also suggested
by Harms and co-workers where they found that fghA mutant
in Paracoccus denitrificans was able to grow on choline, a
formaldehyde-generating substrate, which comes in agree-
ment with our results [6]. Interestingly, during our bioinfor-
matic analysis, we found that the two E. coli proteins YeiG
and FrmB are two homologs for FghA of M. catarrhalis
O35E. Gonzalez and co-workers mentioned that the simul-
taneous deletion of both yeiG and frmB genes is required
to increase the sensitivity to formaldehyde, since the two
proteins contribute to the detoxification of formaldehyde [7].
This could be the case in M. catarrhalis, with a structural
homolog to FghA rather than a sequence homolog that is yet
to be discovered, as none could be found in the proteome
of M. catarrhalis using blast analysis. A study conducted
by Potter and co-workers reported that each of the single
mutants ΔestD and ΔadhC showed identical zones of inhi-
bition to that of the WT by the disc diffusion formaldehyde
sensitivity assay [17]. In the current work, the disc diffusion
method could not differentiate the susceptibilities of the con-
structed mutants and their wild-type counterpart. However,
upon using more quantitative methods, the differences in
the formaldehyde susceptibilities were more prominent. It
was found that a significant difference exists between the
adhC–fghA mutant tested compared to the wild type which
demonstrated that the system contributes to formaldehyde
detoxification.
Previous studies have revealed the important role played
by the adhC–fghA system in bacterial virulence [17, 20].
In the current work, both the ΔfghA and the ΔadhC-fghA
mutants show marked decreased fitness in a pulmonary
clearance model as is evidenced by the significantly lower
colony counts retrieved from mice infected with these
strains. This shows that regardless of its direct role in for-
maldehyde detoxification, FghA could contribute to the
resistance to clearance of M. catarrhalis from the host cells
by a mechanism that is yet to be elucidated. Interestingly,
the obtained data come in accordance with a study which
showed that an FghA homolog, EstD in N. gonorrhoeae, is
necessary for bacterial growth in the host’s cervical epithe-
lial cells, although it did not show a formaldehyde-sensitive
phenotype using disc diffusion assay. The mentioned study
reported that EstD had a potential role in the nitrosative
stress defense system ofN. gonorrhoeae which allows it to
counteract the killing effect of nitric oxide [9] released by
phagocytic cells in the inflammatory response to infection
[17]. This shows that FghA could be involved in combat-
ing other kinds of stresses; hence, its role in pathogenesis
warrants future studies. The significant increase in the pul-
monary clearance extent of ΔfghA as well as ΔadhC-fghA
compared to WT O35E reveals the necessity of both genes
for survival in the respiratory tract of the host. Moreover, the
current findings are similar to a previous study that pointed
out to the possible important role the AdhC–EstD system
plays in the survival and virulence of N. meningitidis, after
observing that the ΔadhC–estD mutant is non-viable in
experimentally induced biofilms [20].
To put this in a more biologically relevant context, Chen
and co-workers reported that the concentration of formal-
dehyde in the blood of healthy individuals is estimated to
be around 0.1mM [4]. However, it is assumed that dur-
ing inflammation, infection, and the associated respiratory
burst, the localized concentration of formaldehyde would
rise above the normal non-toxic levels. Furthermore, the
Fig. 4 Loss of the adhC–fghA operon and fghA results in increased
pulmonary clearance of M. catarrhalis in the murine pulmo-
nary infection model. A box plot representing the colony counts
in log10 CFU/mL, of M. catarrhalis WT O35E, ΔfghA, ΔfghA/R,
ΔadhC-fghA double mutant, and ΔadhC-fghA/R as obtained from
the lungs of infected mice. The bars span the difference between the
minimum and maximum readings. The + sign represents the mean of
the log10CFU/mL. The graphs were generated using GraphPad prism
version 9.0.0 (GraphPad Software, San Diego, CA, USA). Statisti-
cal analysis of data was performed by one-way ANOVA in GrapPad
Prism. The * indicates that the difference is statistically significant
as determined by Tukey’s multiple comparison test (**P ≤ 0.01),
(***P ≤ 0.001), and (****P ≤ 0.0001)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Medical Microbiology and Immunology (2024) 213:3 Page 9 of 10 3
complexity of the host response on the molecular level
would certainly mean that other stresses are present and
could contribute to the significantly higher clearance
observed with the mutants.
Conclusion
This study reports for the first time anfghA mutant and an
adhC/fghA double mutantphenotypes in the emerging path-
ogenM. catarrhalis.The findings in this research indicate
that the system plays a crucial role in formaldehyde detoxifi-
cation and in lung colonization by M. catarrhalis. Moreover,
these findings shed light on the importance of understanding
the adhC–fghA system inM. catarrhalisto help in the poten-
tial development of novel therapeutics to combat infections
caused by this emerging drug-resistant pathogen.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00430- 024- 00785-0.
Author contributions All authors have contributed to the conceptu-
alization of the idea. D.O, N.M.E, and A.S.A have contributed to the
methodology and data curation. All authors have contributed to the
analysis and interpretation of the data. D.O and N.M.E. have contrib-
uted to the preparation of the original draft: All authors have read,
revised, and approved the final version of the manuscript.
Funding Open access funding provided by The Science, Technology &
Innovation Funding Authority (STDF) in cooperation with The Egyp-
tian Knowledge Bank (EKB). No funding was received for conducting
this study.
Declarations
Conflict of interest The authors have no relevant financial or non-fi-
nancial interests to disclose.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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