4 | Discussion
K. variicola is an emerging human pathogen with increasing
antimicrobial resistance and virulence, which poses a threat to public
health [1]. CRISPR/Cas system is an acquired immunity system, the
most abundant form of innate immunity in prokaryotes. However, the
diversity and characterization about CRISPR/Cas system in K.
variicola is not well known. Similar to K. pneumoniae [28],K. variicola strains
harbored three types of CRISPR/Cas systems, including type I-E, type
I-E*, and type Ⅳ-A. Indeed, K. variicola was a member of theK. pneumoniae complex, which phylogenetically related to K.
pneumoniae [6, 16, 35]. The consistency of CRISPR types provides
new evidence for their evolutionary relationship.
Multiple types of CRISPR/Cas systems coexisted in the same strain inK. variicola . The coexistence of type I-E and IV-A system was
found in five strains, and type I-E* and IV-A system coexisted in one
strain. An explanation for this
phenomenon was that the coexistence of different types of CRISPR/Cas
systems contribute to exploiting mutual cas genes to perform
corresponding functions [36]. In this study, all type IV-A systems
lacked cas1 and cas2 genes. cas1 and cas2genes are indispensable in the acquisition of spacers during adaptation
stage [37]. It could be speculated that type IV-A system can utilizecas genes from type I system to perform adaptation roles.
Meanwhile, we also noted that the PAM sequences of type I-E and type
IV-A systems were identical, which further affirms their functional
relationships. Additionally, it has been demonstrated that the synergy
between coexisting subtypes was more conducive to avoiding immune
evasion of MGEs [38, 39]. One study has found that type III
CRISPR/Cas systems could provide redundancy to overcome phage escape
from type I systems [38]. Accordingly, the coexistence of type I and
type IV-A system in K. variicola reinforces the immune defenses
against MGEs. In addition, the absence of cas2 and cas3were found in type I-E CRISPR/Cas system. During CRISPR adaptation, the
Cas2 dimer functions as an adaptor protein and forms a binding surface
for the protospacer DNA combined with two Cas1 dimers [40]. Thecas3 harbors both nuclease and helicase activities, which is
responsible for cutting and degrading invading DNA [41]. The
different degree absence of cas2 and cas3 genes in type
I-E system may represent a degenerated adaptation immunity role.
MLST has been applied to infer the phylogenetic relationship of strains
[42]. Here, we observed the strong association between the
distribution of chromosome-encoded type I CRISPR systems and MLST inK. variicola , which was also found in K. pneumoniae[43]. Nevertheless, plasmid-borne type IV-A systems in K.
variicola were randomly
distributed across MLST. This phenomenon could be attributed to the
stable inheritance of chromosome-encoded CRISPR systems to the offspring
and the HGT of CRISPR/Cas system mediated by plasmid. Moreover, it has
been demonstrated that the scarcity of chromosome-encoded type Ⅰ systems
contributes to the global expansion of multidrug-resistant ST11 and
ST258 clones in K. pneumoniae [44]. It can be speculated that
the uneven distribution of CRISPR systems may be a contributor to the
accumulation of ARGs in specific lineages of K. variicola . It has
been reported that CRISPR-positive plasmids were rich in ARGs inKlebsiella genus [29]. The adaptive immune function carried
by CRISPR might confer drug-resistant plasmids more competitive
advantage in survival.
Although different CRISPR loci
contain diverse repeat sequences, the base composition and arrangement
of repeat sequence are conserved in the same CRISPR/Cas types [45].
We also noticed that the repeat sequences showed conservation within the
same subtypes in K. variicola . However, there were a small number
of base differences in the repeat sequences of the same CRISPR/Cas
system. Although the repeat sequences were diverse during evolution, the
function was conserved. The repeat sequences could be transcribed during
expression stage, thereby forming a stable RNA secondary structure
[45, 46]. In this study, all types of repeat sequence formed
relatively stable secondary structure. In secondary structures,
conserved motifs interact to generate “stem-loop” that act as
processing points for pre-crRNA through the cas6 endonuclease
[45-47]. According to MFE value theory, stems with higher GC content
and larger match base numbers tend to harbor lower MFE, suggesting more
stable secondary structure. Here, the MFE of the type I-E repeat
sequence was the smallest, representing the most stable RNA secondary
structure.
The adaptation activity of CRISPR/Cas system can be measured by the
number of the spacer [22]. The number of the spacers in K.
variicola was diverse for different CRISPR/Cas types, ranging from 3 to
41 spacers, which indicated their different abilities to obtain spacers.
Type I-E system contained the most spacers, thereby demonstrating its
strong adaptation ability. The protospacer sequence was the specific
sequence on the foreign gene that was consistent with spacers [48,
49]. About one-third of spacers in K. variicola were homologous
to plasmids or phages. The proportion of K. variicola spacers
targeting foreign genetic elements was significantly higher than other
bacteria, such as Staphylococcus [50] and Clostridium
perfringens [51]. It might be owing to that the sequencing ofK. variicola -related MGEs was overrepresented in the current
databases. In this study, type I-E spacers showed homology with plasmids
more than phages. Previous studies have shown that type I-E CRISPR/Cas
systems in K. pneumoniae affected the dissemination of IncF
antibiotic-resistant plasmids [44, 52]. Considering the evolution
relationship between K. variicola and K. pneumoniae , it is
possible that type I-E systems in K. variicola play a similar
role in limiting the uptake and survival of antibiotic-resistant
plasmids. Different from type I-E system, type I-E* system biasedly
targeted phages, suggesting their different responsiveness to diverse
MGEs. Uniquely, plasmid-borne type
IV-A spacers target plasmids more effectively than phages. Moreover, the
target preference for plasmids in type IV-A system was more obvious than
chromosome-encoded type I system (type I-E and I-E*). The strong
association between type IV-A system and plasmids can be attributed to
the fierce battle between similar entities competing for overlapping
niches and resources [53-55].
The spacer sequence reflects the history of interaction between bacteria
and MGEs [56]. We observed that K. variicola spacers prefer
to target MGEs from K. pneumoniae, which suggested that K.
varricolla were frequently challenged by K. penumoniae MGEs.
Previous study has shown that K. variicola displayed similar
biological features to K. pneumoniae [57].
The
strong interaction between K. varricola and K.
pneumoniae that are implied by spacer target events provides new
evidence for their evolutionary affinity. In addition, we analyzed the
genes-targeted characteristics. The diversity of coding products
indicated the wide range of spacer sources and the diversity of CRISPR
functions [58]. Conjugative
transfer-related proteins were the most frequently targeted proteins,
such as TrbI, TrbD, and TrbH. These Trb proteins were required for pilus
extension and conjugal DNA transfer, which were relatively active
[59-61].
Conjugative transfer was the main
mechanism in the spread of ARGs in which conjugative transfer proteins
played an important role in mediating plasmid DNA exchange [62]. In
our study, we observed that 15.15% (35/231) spacers targeted
conjugative transfer proteins, which further highlight the important
role of CRISPR/Cas system in influencing the dissemination of antibiotic
resistance. In addition, conserved genes encoding phage tail protein,
methyltransferase, kinase, terminase were also frequently targeted by
CRISPR spacers. These direct targets to conserved gene regions is
beneficial for improving the defense efficiency of CRISPR systems and
avoiding immunity escape of MGEs.
In conclusion, our results discovers
that K. variicola harbors diverse and complex CRISPR/Cas systems,
including three types. The distribution of chromosome-encoded type I
CRISPR/Cas system is associated with MLST whereas plasmid-encoded IV-A
system is randomly distributed across MLST. Spacer homology analysis
reveal that CRISPR/Cas systems targeted diverse MGEs, especially
conjugative transfer-related proteins related to antibiotic-resistant
plasmids, thereby suggesting the critical role of CRISPR/Cas system in
control ARG spread in K. variicola . Overall, this study provides
new insights into the evolution of CRISPR/Cas systems in this specie.