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Title: Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation}
Article:
Abstract:
Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species( ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis( Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair( MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC- mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC- mice was significantly higher than that in NAC+ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms.
Introduction:
Vibrio cholerae, the etiological agent of the pandemic disease cholera, resides in aquatic environments and can also colonize human intestines following ingestion of contaminated food and water. In order to survive in both aquatic and host environments, V. cholerae has the ability to cope with harsh conditions during the transition to the host gut and during subsequent growth[1]. For example, upon infection, V. cholerae senses host signals and is able to coordinate both virulence gene activation and repression to evade host defenses and successfully colonize intestines[2–5]. Late in the infection, V. cholerae also optimally modulates its genetic programs for the forthcoming dissemination into the aquatic environment[6, 7] where it is often associated with abiotic or biotic surfaces such as phytoplankton and zooplankton. These associations enable the formation of biofilms, which provide protection from a number of environmental stresses; including nutrient limitation, protozoa predation, and bacteriophage infection[8]. Additionally, biofilms may enhance infectivity due to their acid-resistant properties and higher growth rate during infection[9, 10]. One of the major stresses V. cholerae must overcome is exposure to reactive radical species. Reactive compounds, including reactive oxygen species( ROS), are abundant in marine systems[11]. V. cholerae also encounters oxidative stress during the later stages of infection, as demonstrated by an increase in ROS levels and a decrease in the levels of host antioxidant enzymes during V. cholerae-induced diarrhea[12, 13]. It has been previously demonstrated that catalases( KatG and KatB), peroxiredoxin( PrxA), organic hydroperoxide resistance protein( OhrA), a redox-regulated chaperone( Hsp33), and a DNA-binding protein from starved cells( DPS) are important for V. cholerae ROS resistance[14–17]. ROS resistance in V. cholerae is known to be tightly regulated through a variety of mechanisms. OxyR is required to activate catalase genes and dps, and is modulated by another OxyR homolog, OxyR2[14, 16, 18]. Quorum sensing systems[19], PhoB/PhoR two-component systems[20], and the virulence regulator, AphB, also play important roles in oxidative stress response[21]. Further identifying bacterial stress responses to host-derived ROS is important for understanding V. cholerae pathogenesis. In this study, we used Tn-seq to screen for V. cholerae genes that are involved in ROS resistance during infection. By comparing colonization in control mice to mice treated with antioxidant N-acetyl cysteine( NAC) that reduces the production of ROS in murine intestines[15], we found that deletion of mutS, encoding a key component in the DNA methyl-directed mismatch repair( MMR) system, results in a significant colonization advantage compared to wildtype in ROS-rich mice. The MMR system is highly conserved from bacteria to humans and is critical for maintaining the overall stability of the genetic material[22]. Mutations in this pathway lead to hypermutation rates across the genome. It has been shown that inactivation of the MMR system of various bacterial pathogens, such as Escherichia coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa leads to better adaptation and persistence of these pathogens in murine models[23–26]. It has been proposed that under certain stressful conditions, hypermutators are selected in the total population by hitchhiking with the adaptive mutations that they produce. However, the mechanism( s) by which hypermutators become better persistors is less clear. In this work, we developed a strategy to study bacterial temporal hypermutation in vivo and found that mutations resulting in increased catalase production and increased biofilm formation, demonstrated by rugose colony phenotypes, may lead V. cholerae hypermutators to display colonization advantages in ROS-rich mouse intestines.
Discussion:
Bacterial pathogens are constantly confronted with changing and aggravating environments and have been known to leverage genetic adaptation as a means to overcome challenges faced in these environments. In this study, we used a streptomycin-treated mouse model to study V. cholerae ROS resistance in vivo. Bacteria experience host-generated oxidative stress in the streptomycin-treated adult mouse model[15, 27, 28]. Inclusion of antioxidant N-acetyl cysteine( NAC) significantly reduced ROS levels[15]( Fig G in S1 Text). For mice without the streptomycin treatment, ROS levels were lower than streptomycin-treated mice, but remained detectable( Fig G( A) in S1 Text). In addition, it has been reported that during choleric diarrhea, ROS levels were increased in the host[12, 13]. Taken together, it is suggestive that ROS stress encountered by V. cholerae in the streptomycin-treated mouse model may be physiologically relevant. By the Tn-seq screen, we discovered that hypermutation rates resulting from the impairment of the V. cholerae mismatch repair system( ΔmutS) led to a colonization advantage in mice, which was not observed in NAC-treated mice. E. coli colonization studies of mouse intestines have shown that hypermutation is initially beneficial because it allows for a rapid adaptation to the mouse gut environment[26]. However, such strains then experience a loss of fitness due to the constant accumulation of detrimental mutations. To prevent additional detrimental mutations and to be able to study those mutations that conferred a colonization advantage in vivo, we complemented ΔmutS isolates from NAC- mice immediately after isolation. Further study shows that passage of ΔmutS through NAC- mice resulted in the enrichment of catalase-overproducing isolates and a high frequency rugose phenotype. These ΔmutS* isolates remained super-colonizers in NAC- mice but did not gain advantages in NAC+ mice( Fig 3A). We also examined infant mouse colonization( Fig H( A) in S1 Text) as well as virulence gene expression( Fig H( B&C) in S1 Text) and found that compared to wildtype, some ΔmutS* isolates were defective in infant mouse colonization and virulence factor production. These results suggest that mutations are specifically selected to overcome ROS stress in the NAC- mice. Indeed, in a previous report[45] by Davies, et al., it was observed that V. cholerae ΔmutS mutants displayed an approximately 5-fold defect in infant mouse colonization. Considering the short incubation time in infant mouse colonization( 18 hrs) and the speculated lack of inflammation in infant mouse gut, it is possible that ΔmutS mutants do not experience the same selective pressures as in ROS-rich adult mice. Similarly, in P. aeruginosa, ΔmutS mutants are attenuated in a mouse model of acute infections but are favored in long term persistence of oropharyngeal colonization in cystic fibrosis mice[25]. Many hyper-mutational bacterial pathogens are frequently identified from clinical and environmental isolates, including E. coli, Salmonella, P. aeruginosa, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae[46]. This is often the case when bacteria need to adapt a new stressful environment. For example, a high percentage of mutators of P. aeruginosa, H. influenzae, and S. aureus were isolated from cystic fibrosis patients who received antibiotic treatments[47]. Infection of a mammalian host is certainly another new environment to adapt to and an increase in genetic variability can help to cope with host defense systems[48]. V. cholerae hypermutators have also been found in clinical isolates. In a recent study[49], Didelot et al. reported that among 260 V. cholerae genomes they sequenced and analyzed, 17 isolates have unusually high number of SNPs that are evenly spread throughout their genomes. Further analysis shows that 14 of these 17 genomes possess genetic variations in one or more of four genes in the MMR system and the mutation rate of these strains are significantly increased compared to the others. Interestingly, the majority of these hypermutator strains were isolated between 1961 and 1965, relatively soon after the beginning of the seventh pandemic. The authors cautiously speculated that hypermutators might be causally associated with the rapid spread of the seventh pandemic. In addition, a mobile element is found to insert into the mutS gene of a marine Vibrio species, providing a new mechanism for altering the mutation rate[50]. Hypermutation may promote adaptive evolution for bacteria, but the high mutation rate comes at a cost in fitness in the long term[26]. It has been proposed that bacteria may transiently modulate their mutation rates to balance the trade-off between adaption and the accumulation of detrimental mutations[51]. For example, the expression of mutS is downregulated by RpoS in response to antibiotic stress, which increases the mutation rate in several bacterial species including V. cholerae[52]. In Streptococcus pyogenes, the integration and excision of a prophage inserted between mutS and mutL causes a reversible increase in mutation rate in response to the environmental stress[53]. We found that in wildtype V. cholerae, mutation rate was significantly increased when colonizing NAC- mice compared to NAC+ mice( Fig 6A). This finding suggests that V. cholerae might utilize increased mutation rates as a temporal strategy for adopting advantageous phenotypes during infection of a ROS-rich host. Interestingly, our Tn-seq screen( S1 Data) revealed that rpoS mutants failed to colonize NAC- mice but not NAC+ mice. Further testing is required to investigate whether the observed increase in mutation rate in NAC- mice is RpoS-mediated. Moreover, the rugose variants isolated from NAC- mice could reverse to smooth colonies in vitro( Fig 4C), indicative of use of temporal adaptive strategies by V. cholerae to combat ROS during infection. Of note, the mechanism of V. cholerae smooth-rugose phase variation is not clear, but DNA repair pathways have been implicated in phase variation in several species, including Neisseria gonorrhoeae, V. parahaemolyticus, and Pseudomonas sp.[54–57]. Interestingly, the high reversion rate from rugose to smooth colonies under aerobic growth( Fig 4C) occurred in mutS complemented background( ΔmutS*), suggesting that reversion is not due to DNA mutation. Cell variants in some bacterial species are generated without the burden of mutation, but rather from reverse biostability, which can be controlled by genetic mechanisms such as DNA rearrangement or epigenetic mechanisms such as DNA methylation[58, 59]. Alternatively, the rapid reversion from rugose to smooth in vitro even though these cells have been repaired for mutS may simply because that the selective pressure for reversion to the smooth variant is remarkably strong during aerobic growth and therefore the reverting mutations arise rapidly. The exact mechanisms of O2-dependent rugose-to-smooth phenotypic switch is currently under investigation. Hypermutable strains are often associated with higher incidences of antibiotic resistance than strains with lower mutations rates. This study proposes a model of in vivo temporal hypermutation by mutating MMR and complementing mutants with functional MMR after isolation. This approach allowed for the identification of ROS resistance mechanisms that could be genetically upregulated under ROS stress. It is likely that this approach could be utilized in the context of distinct stressors such as low pH, desiccation, nitrosative stress, etc., revealing likely mechanisms used to overcome those specific environments by comparing mutation spectra or phenotypic changes between experimental groups. In reverse order, MMR mutants could also be used to shed light on stresses experienced in undefined environments by bacteria by associating enriched pathways with stressors. Insight into the mechanisms used to overcome specific stressors could be used to refine antibacterial strategies. This insight would allow for the proactive targeting of arising mutators under treatment, preventing resistant lineages. This application could improve the efficacy of antibacterial agents and reduce the incidence of resistant mutators.
Cholera is a devastating diarrheal disease that is still endemic to many developing nations , with the worst outbreak in history having occurred recently in Yemen . Vibrio cholerae , the causative agent of cholera , transitions from aquatic reservoirs to the human gastrointestinal tract , where it expresses virulence factors to facilitate colonization of the small intestines and to combat host innate immune effectors , such as reactive oxygen species ( ROS ) . We applied a genome-wide transposon screen ( Tn-seq ) and identified that deletion of mutS , which is part of DNA mismatch repair system , drastically increased colonization in ROS-rich mice . The deletion of mutS led to the accumulation of catalase-overproducing mutants and a high frequency rugose phenotype when exposed to ROS selective pressures in vivo . Additionally , ROS elevated mutation frequency in wildtype , both in vitro and in vivo . Our data imply that V . cholerae may modulate mutation frequency as a temporal adaptive strategy to overcome oxidative stress and to enhance infectivity .