The adcA and lmb Genes Play an Important Role in Drug Resistance and Full Virulence of Streptococcus suis

ABSTRACT Streptococcus suis is an recognized zoonotic pathogen of swine and severely threatens human health. Zinc is the second most abundant transition metal in biological systems. Here, we investigated the contribution of zinc to the drug resistance and pathogenesis of S. suis. We knocked out the genes of AdcACB and Lmb, two Zn-binding lipoproteins. Compared to the wild-type strain, we found that the survival rate of this double-mutant strain (ΔadcAΔlmb) was reduced in Zinc-limited medium, but not in Zinc-supplemented medium. Additionally, phenotypic experiments showed that the ΔadcAΔlmb strain displayed impaired adhesion to and invasion of cells, biofilm formation, and tolerance of cell envelope-targeting antibiotics. In a murine infection model, deletion of the adcA and lmb genes in S. suis resulted in a significant decrease in strain virulence, including survival rate, tissue bacterial load, inflammatory cytokine levels, and histopathological damage. These findings show that AdcA and Lmb are important for biofilm formation, drug resistance, and virulence in S. suis. IMPORTANCE Transition metals are important micronutrients for bacterial growth. Zn is necessary for the catalytic activity and structural integrity of various metalloproteins involved in bacterial pathogenic processes. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. Thus, pathogenic bacteria must acquire Zn during infection in order to successfully survive and multiply. The host uses nutritional immunity to limit the uptake of Zn by the invading bacteria. The bacterium uses a set of high-affinity Zn uptake systems to overcome this host metal restriction. Here, we identified two Zn uptake transporters in S. suis, AdcA and Lmb, by bioinformatics analysis and found that an adcA and lmb double-mutant strain could not grow in Zn-deficient medium and was more sensitive to cell envelope-targeting antibiotics. It is worth noting that the Zn uptake system is essential for biofilm formation, drug resistance, and virulence in S. suis. The Zn uptake system is expected to be a target for the development of novel antimicrobial therapies.

space between the genus name and the specie. line 22: adcA or adcACB mutant strain ? line 23: Zinc-limited , in lowercase characters line 42 and 78: AdcA and Lmb are zinc-binding proteins and not transporters as the complete transporter also comprise the integral membrane protein, AdcB, a nucleotide binding domain, AdcC and histidine triad (HT) proteins (Plumptre et al. 2014;Moulin et al. 2019) line 44, 128, 429: sensitivity or susceptibility and not sensitive line 77: Plumptre et al. 2014 andMoulin et al. 2019 also highlighted two distinct routes of zinc acquisition in Streptococci mediated by either AdcA alone, or via Lmb/AdcAII in concert with the Sht-family proteins. These modes of zinc acquisition have been attributed to the distinct structural features of AdcA and AdcAII. This should be mentioned here and used to describe more precisely AdcA and Lmb of S. suis. line 121: the sentence has no sense Line 271-276 and figure 2: Should be removed from the results section and place in material and methods. Line 275: This is the first figure, so that it should be numbered as figure 1, etc... Line 279: a part of the sentence is missing Fig 5A and B: Both complementation strains do not fully complement. How do you explain this? Moreover, in TSB medium, in which biofilm experiments were conduced, both lmb and adcA gene are repressed because of the zinc. This should be at least discussed. Fig. 5C: there is no mention of the growth conditions for the q-RT PCR experiments: in which medium ? At which phase of growth? line 328-330: to my knowledge none of the genes that were tested here is directly linked to adhesion. For example CcpA and PerR are transcriptional regulators controlling respectively carbon metabolism and metal homeostasis and can't be named "adhesion genes". Did they test others real "adhesion gene", like gene encoding pili ? In Fig.5, most of the differences are below a 2-fold change. I am not convinced by the biological relevance of these differences. Moreover the title "AdcA and Lmb contribute to biofilm formation in S. suis by downregulating the expression of adhesion genes" is not supported by the data. There is no evidence that the difference in perR and gdh expression has any connection with the difference in biofilm formation. Additional experiments are needed to prove it. Fig. 8: Unfortunately, similar survival curves and bacterial counts in organs have been published in June 2022 by Zheng et al.. Line 349: remove "was" in the sentence Line 388 "AdcA is associated with direct recruitment of Zn " : in which bacteria ? Reference ? Line 389 "The lmb gene encodes a laminin, which has previously been associated with Zn acquisition". Lmb certainly not encodes a laminin ! It is a laminin-binding protein. Reference ? Line 394 "The regulation mechanism of Zn transport in S. suis needs to be further elucidated": in numerous Streptococcal species, the Adc/lmb transporter is regulated by AdcR (Shafeeq et al. 2011 ;Sanson et al. 2015 ;Moulin et al. 2016). This is certainly also the case in S. suis and it should be discussed here. Line 417: "nutrient availability" rather than "hunger resistance" Line 428: The authors did not provide any reference linking the tested genes with adhesion. Moreover their data do not show that the reduced biofilm formation is due to the downregulation of these genes.
Reviewer #2 (Comments for the Author): The manuscript by Mingzheng Peng et al. provides a systematic analysis of the phenotype displayed by knock-out mutants of the genes adcA and lmb, coding for the two Zn transporters in Streptococcus suis. The manuscript presents a useful study on the influence of zinc homeostasis in cell adhesion, invasion and biofilm formation by S. suis as well as on its tolerance towards antibiotics. However, a more detailed genetic characterization of the strain used should be reported and discussed as the manuscript suffers from the lack of a thorough presentation and analysis of the data. For instance, there is no mention of the source of the strain used in the study or reference to its genome sequence, which has been present in the NCBI database since 2017. Furthermore, a clear comprehension of the work is hindered by the poor quality of the English language usage and punctuation. In addition, the inaccurate use of technical terminology renders the manuscript more difficult to follow. For example: line 273 "complementary strains" instead of "complementation strains" line 279 strain in TSB. To explore there should be a comma instead of a full stop I strongly suggest that the manuscript should be checked by an English-speaking colleague, the clarity of the topics presented would benefit significantly. In conclusion, the manuscript offers an extensive characterisation of the effects of loss of zinc homeostasis in S. suis, but the authors must capitalise better the considerable experimental work by presenting their study in a more careful manner and provide a more complete discussion of their results compared to the many relevant reports available in the literature.

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The manuscript by Mingzheng Peng et al. provides a systematic analysis of the phenotype displayed by knock-out mutants of the genes adcA and lmb, coding for the two Zn transporters in Streptococcus suis.
The manuscript presents a useful study on the influence of zinc homeostasis in cell adhesion, invasion and biofilm formation by S. suis as well as on its tolerance towards antibiotics. However, a more detailed genetic characterization of the strain used should be reported and discussed as the manuscript suffers from the lack of a thorough presentation and analysis of the data. For instance, there is no mention of the source of the strain used in the study or reference to its genome sequence, which has been present in the NCBI database since 2017.
Furthermore, a clear comprehension of the work is hindered by the poor quality of the English language usage and punctuation. In addition, the inaccurate use of technical terminology renders the manuscript more difficult to follow. I strongly suggest that the manuscript should be checked by an English-speaking colleague, the clarity of the topics presented would benefit significantly.
In conclusion, the manuscript offers an extensive characterisation of the effects of loss of zinc homeostasis in S. suis, but the authors must capitalise better the considerable experimental work by presenting their study in a more careful manner and provide a more complete discussion of their results compared to the many relevant reports available in the literature.

Response to Reviewers
We would like to express our sincere thanks to the reviewers for their constructive comments and suggestions, which help us in depth to improve the quality of the manuscript. We have tried our best to revise the manuscript according to the comments. The following response is point-by-point towards the reviewers' comments Reviewer comments: Reviewer #1 (Comments for the Author): This manuscript describes the role of AdcA and Lmb, two zinc-binding proteins in Streptococcus suis. Although this study is rather complete in term of methodologies, a large part of the results were already published (see "The AdcR-regulated AdcA and AdcAII contribute additively to zinc acquisition and virulence in Streptococcus suis", Zheng et al. 2022). I also have concerns concerning some data, in particular with the results on biofilms (see below). Finally the english is sometimes of poor quality, there is a lack of references and a lot of mistakes which requires a more careful re-reading of the manuscript. You can find below some suggestions that should help the authors to improve their manuscript. RE: Thank you for your nice suggestion.
(1)Line 19, 33, 70, 87, 90, 304, 338, etc...: A bacteria name must be in italic with a capital letter for the genus name and with a space between the genus name and the specie. RE: Thank you very much for your reminder. We have fixed these corresponding errors in the revised manuscript. Please refer to line 19,32,70,88,90,93,96,99,310 and 344 in the revised manuscript.
(2)line 22: adcA or adcACB mutant strain ? RE: Thank very much for your advice. We constructed an adcA deficient strain in this study, we have replaced adcACB with adcA in the revised manuscript. Please refer to lines 22 in the revised manuscript.
(3)line 23: Zinc-limited , in lowercase characters RE: Thank you for your nice suggestion. We have replaced Zinc-limited with zinc-limited in the revised manuscript. Please refer to lines 24 in the revised manuscript.   (10)Line 279: a part of the sentence is missing. RE: Thank you for your nice suggestion. We have replaced this sentence with "The transcription levels of adcA and lmb in CDM medium with or without Zn were tested to explore the role of AdcA and Lmb of S. suis in Zn-restricted environments." in the revised manuscript. Please refer to lines 286-287 in the revised manuscript. .
(11) Fig 5A and B: Both complementation strains do not fully complement. How do you explain this? RE: Thank you for your nice suggestion. We repeated the biofilm experiment several times with the same results. As we complement the gene by transferring plasmid. We found that the expression level of related genes in the complement strain was lower than that in the wild strain by RT-PCR, which might explain that both complementation strains do not fully complement.
(12)Moreover, in TSB medium, in which biofilm experiments were conduced, both lmb and adcA gene are repressed because of the zinc. This should be at least discussed. RE: Thank you for your nice suggestion. It has been reported that zinc ions are important for biofilm formation (Lam et al., 2022) (Li et al., 2022. In the previous experiment, we found that there was no significant difference in the growth of wild strains and mutant strains in TSB medium. Although the medium contains a certain concentration of zinc ions, the bacterial intake of zinc ions from the environment is reduced due to the absence of zinc uptake proteins AdcA and Lmb, thus affecting biofilm formation. We added this content to the discussion section of the article. Please refer to lines 443-449 in the revised manuscript. (13) Fig. 5C: there is no mention of the growth conditions for the q-RT PCR experiments: in which medium ? At which phase of growth? RE: Thank you for your nice suggestion. The WT or ∆adcA∆lmb strains were grown in TSBS medium overnight, then washed three times with PBS, overnight bacterial culture was diluted 1:100 with fresh TSB and grown until the OD600 reached 0.6. Then the total RNA of bacteria was extracted.We have added this condition in the revised manuscript. Please refer to lines 3-4 in the figure 5.
(14)line 328-330: to my knowledge none of the genes that were tested here is directly linked to adhesion. For example CcpA and PerR are transcriptional regulators controlling respectively carbon metabolism and metal homeostasis and can't be named "adhesion genes". Did they test others real "adhesion gene", like gene encoding pili ? RE: Thank you for your Correction. We have changed this part into "AdcA and Lmb contribute to biofilm formation of S. suis by influencing bacterial virulence" in the revised manuscript. It has been reported that biofilm formation is related to the bacterial virulence (Lam et al., 2022) (Wang et al., 2019b. We repeated the q-RT PCR experiment of eight virulence-related genes (gapdH, srtA, gdpP, perR,gdh, gor, ccp and fbps) of S. suis and updated the results. ( Liu B et al., 2020;Wang Y et al., 2019;Vega et al., 2016;Gao et al., 2013;Zhang et al., 2006).Of these genes, gor and gdh were down-regulated significantly. Studies have suggested that gor gene is involved in the synthesis of glutathione reductase, which has been implicated in oral L. monocytogenes infections in mice (Mansfield and Mansfield, 2007). Glutamate dehydrogenase (GDH) is a key enzyme that connects carbon and nitrogen metabolism. It is a very important functional molecule in the energy metabolism process of bacteria, and plays a significant role in pathogenicity，which contributes to the virulence of S. suis type 2 (Okwumabua and Chinnapapakkagari, 2005) Although it has been confirmed that AdcA and Lmb can regulate the expression of multiple genes involved in regulating bacterial virulence, the specific regulatory pathway is unknown. In the future, it would be interesting to further explore the mechanism of transcription regulation by studying the gene expression of the entire genomes of WT and knockout mutants. We have added this content to the discussion section of the article. Please refer to lines 450-463 in the revised manuscript.
(15)In Fig.5, most of the differences are below a 2-fold change. I am not convinced by the biological relevance of these differences. Moreover the title "AdcA and Lmb contribute to biofilm formation in S. suis by downregulating the expression of adhesion genes" is not supported by the data. There is no evidence that the difference in perR and gdh expression has any connection with the difference in biofilm formation. Additional experiments are needed to prove it. RE: Thank you for your advice. AdcA and Lmb can not directly regulate the expression of corresponding genes. We have changed the title into "AdcA and Lmb contribute to biofilm formation in S. suis by influencing bacterial virulence" in the revised manuscript.We repeated the q-RT PCR experiment and updated the results. You can refer to the question14 for instructions.
(16) Fig. 8: Unfortunately, similar survival curves and bacterial counts in organs have been published in June 2022 by Zheng et al..

RE:
Thank you for your nice suggestion. We have done animal experiment on mice before that, there are two differences between our assays. First, besides WT and Δ adcAΔlmb, we also set ΔadcA, CΔadcA, Δlmb and CΔlmb mouse groups. At 48h after infection with 2×10 8 CFU bacteria, we detected bacterial counts. Our results provide evidence that individual adcA and lmb genes affect pathogenicity of S. suis. which also echoed our previous hypothesis that adcA may have synergistic effects with lmb. Besides, we detected bacterial counts in the blood at different time points (3h, 6h, 9h and 12h), which provided more detailed and comprehensive data about early stage of invasive infections of S. suis (Liu L et al., 2006).
(17)Line 349: remove "was" in the sentence RE: Thank you for your nice suggestion. We have removed "was" in the sentence in the revised manuscript. Please refer to lines 355 in the revised manuscript.
(18)Line 388 "AdcA is associated with direct recruitment of Zn " : in which bacteria ? Reference ? RE: Thank you for your nice suggestion. We have replaced this sentence with "AdcA is associated with direct recruitment of Zn in most streptococci species (Bayle et al., 2011;Makthal et al., 2017, Makthal et al., 2020.".We added the relevant references in the revised manuscript. Please refer to lines 394-398 in the revised manuscript. (19)Line 389 "The lmb gene encodes a laminin, which has previously been associated with Zn acquisition". Lmb certainly not encodes a laminin ! It is a laminin-binding protein. Reference ? RE: Thank you for your nice suggestion. We have replaced this sentence with "The Lmb is annotated as a laminin, which has previously been associated with Zn acquisition.". The reference (Tedde, V et al., 2016) has indicated this in detail. We added the relevant references in the revised manuscript. Please refer to lines 394-398 in the revised manuscript.
(20)Line 394 "The regulation mechanism of Zn transport in S. suis needs to be further elucidated": in numerous Streptococcal species, the Adc/lmb transporter is regulated by AdcR (Shafeeq et al. 2011 ;Sanson et al. 2015 ;Moulin et al. 2016). This is certainly also the case in S. suis and it should be discussed here. RE: Thank you for your nice suggestion. Group A streptococcus and S. pneumoniae adaptive responses to Zn limitation are coordinated by the Zn-sensing transcription regulator adhesion competence repressor (AdcR) (Makthal and Kumaraswami, 2017;Manzoor et al., 2015;Sanson et al., 2015).