Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies

ABSTRACT To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

established a workflow method to systematically test different compounds and concentrations, resulting in optimized formulations capable of supporting serial passaging and robust growth. The author did present some data to prove his conclusion. However, there are some questions, editing, and formatting errors in the manuscript needed to be addressed. Overall, this article needs further revision before publication. Comments: (1) Page 2, line 33: the abbreviations of "CMRL" should be written in their full names when they first appear.
(2) Page 2, line 35, line 36, line 37, and line 38: I don't think it's necessary to capitalize the first letter of these words (such as Cysteine, Yeastolate, Cysteine...). Please check the full text.
(3) Page 2, line 43: The sentence "Diseases associated with Mycoplasmas are an important economic burden in both human and livestock systems" needs to be refined (4) Page 4, line 69: the abbreviations of "M. pneumoniae" should be written in their full names when they first appear. (5) Page 4, line 77: "BHI (brain heart infusion broth), PPLO (beef heart infusion and peptones)" change to "brain heart infusion broth (BHI), beef heart infusion and peptones (PPLO)" (6) Page 6, line7, line120: "lipids, vitamins and co-factors, essential metals and ...." change to "lipids, vitamins, co-factors, essential metals and...." (7) Page 11, line 302, line 305; page 13 line 366: "96h", "75cm2" and "72h" change to "96 h", "75 cm2" and "72 h". It needs a space before the unit. Please check the full text. (8) In the part of REFERENCES: Please uniform reference journal names: full or abbreviated? Such as "Physiological Reviews", "Mol Syst Biol.". In addition, If the journal name is presented as an abbreviation, add a dot after each abbreviation word, such as "Mol Syst Biol." Change to "Mol. Syst. Biol." Other questions: (1) Page 6, line129-131: "To facilitate medium preparation and reproducibility, we replaced amino acids, bases, vitamins and inorganic salts by RNA and the commercially available CMRL-1066 medium." What is the basis of this change, and do not see the relevant references.
(2) Page 6, line 135: "This method estimates growth by measuring the change of absorbance (ratio 430 and 560 nm).....". What is the principle of this method, and did not see the reference (3) Page 6, line 141-142: "we tested decreased concentrations of HEPES, detecting a measurable change in pH with 50mM HEPES (Fig. S1)." The data did not show the changes in pH indicators? (4) Page 6, line 141-144: The pH indicators in different mediums should change during the culture. You should judge the effect of different mediums by the degree of pH change, not simply by whether the pH index has changed. (5) Page 7, line 151: "......and the analysis of the metabolic map of M.pneumoniae....." Where is this data? (6) Page 12, line 323-325: "these results show a minor impact in gene expression....." Whether this change will also affect the function of subsequent vaccines (7) In this study, a serum-free medium was developed to replace the traditional medium. In the optimization process, whether there are still some natural ingredients in serum cannot be determined by artificial optimization?
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Thank you for submitting your paper to Microbiology Spectrum. We thank reviewer #2 for carefully reading the manuscript and providing constructive feedback to improve our manuscript. Please, see below a detailed pointby-point response to the specific comments and questions.

Comments:
(1) Page 2, line 33: the abbreviations of "CMRL" should be written in their full names when they first appear.
Following the editorial guideline, we tried to avoid abbreviations in the abstract. Instead, we added the abbreviation of CMRL (Connaught Medical Research Laboratories) in page 6, line 131, when it first appears in the introduction.

Please check the full text.
Thank you for the comment. We have corrected this as suggested.
(3) Page 2, line 43: The sentence "Diseases associated with Mycoplasmas are an important economic burden in both human and livestock systems" needs to be refined We have modified this sentence as follows: " Mycoplasma infections have a significant negative impact on both livestock production and human health". The full name of "Mycoplasma pneumoniae" is defined for the first time in page 4, line 62.
Thank you for noting this error. We have now corrected this through the manuscript. The format of the reference section has been corrected following the journal editorial style.

Other questions:
(1) Page 6, line129-131: "To facilitate medium preparation and reproducibility, we replaced amino acids, bases, vitamins and inorganic salts by RNA and the commercially available CMRL-1066 medium." What is the basis of this change, and do not see the relevant references.
We used as a starting formulation the defined medium previously developed in our laboratory (reference Yus et al., 2009). This medium is not capable of supporting robust growth (as stated in page 6, line 121-126) and contains many components, such as amino acids, individual bases, vitamins or inorganic salts that are added separately.
This makes the preparation of the media difficult and can result in lack of reproducibility when it is prepared. Since RNA (to substitute nucleotide precursors) and the CMRL medium contain most of these components (in addition to other cofactors), we decided to use RNA and this commercial medium to substitute many of the components that were added separately in the previous medium. This strategy facilitated the preparation of the medium and its reproducibility, especially when different media formulations are tested. To make this clear, the sentence in page 6, lines 129-131 has been modified as follows (now page 6, lines 129-135): "For this, we first replaced all the amino acids, bases, vitamins and inorganic salts present in the defined medium reported by Yus et al.,(9), by RNA and the CMRL-1066 medium (Connaught Medical Research Laboratories). This commercially available medium contains most of the components described above, avoiding the addition of these components individually, and therefore improving medium preparation and reproducibility".
(2) Page 6, line 135: "This method estimates growth by measuring the change of absorbance (ratio 430 and 560 nm).....". What is the principle of this method, and did not see the reference As stated in page 6, line 134 (now page 6, line 137), the "growth index" method was previously described by Yus et al (2009) and it has become a reference method to estimate growth in mycoplasmas that acidify the medium when it grows like M. pneumoniae. As described by Yus et al (2009), the analysis of the whole absorbance spectrum of the pH indicator used in the medium (phenol red) showed two wavelengths that respond differently to pH, around 430 nm (yellow) and 560 nm (red). The ratio between these two absorbances correlates well with changes in pH produced by the active lactate metabolism of M. pneumoniae when it grows (medium changes from red to yellow). Based on this principle, this method is an indirect but straightforward method to measure growth. The original reference of this method is now indicated in page 6 line 137. Also, to clarify the principle of the method the sentence in page 6 137-142 has been modified as follows: "This method estimates growth by measuring the change of absorbance (growth index= ratio 430 and 560 nm) in the culture medium, and relies on the fact that M. pneumoniae acidifies this medium when it is metabolically active. A decrease of pH in the medium, detected by an increase in the growth index, is therefore an indirect but straightforward method to measure growth (9)." (3) Page 6, line 141-142: "we tested decreased concentrations of HEPES, detecting a measurable change in pH with 50mM HEPES (Fig. S1)." The data did not show the changes in pH indicators?
We totally agree with this reviewer that Fig. S1 does not directly show changes in pH. However, as stated in the previous response, changes in the growth index (that is represented in Fig. S1) correlates with changes in pH. To clarify this, we have modified the sentence as follows (now page 6, line 146): "...we tested decreased concentrations of HEPES, detecting a measurable change in the growth index with 50mM HEPES (Fig. S1) As commented in the previous responses, the pH indicator actually changes during the culture (from red to yellow) due to the acidification of the medium.
Measuring the ratio absorbance of 430 and 560 nm (growth index) is a quantitative method to measure the degree of these pH changes. To illustrate this, below we show A) Typical M. pneumoniae growth experiment done in a 96-well format in a Tecan plate reader, using a pH indicator, phenol red, to monitor growth (changes from red to yellow as the medium acidifies because of fermentation. B) The ratio of the pH indicator (Phenol red) absorbance at 430 versus 560 nm ("growth index") correlates well with the pH, at the pH range of the experiments performed (5.7-7.7). Equation for calculation of pH from the growth index, estimated for the pH range for which there is linear relationship (pH 5.7-7.7, R2=0.9931). Where is this data?
The analysis and refinement of the metabolic network of M. pneumoniae has been previously published. The data is available in references (9) and (26)   Whether this change will also affect the function of subsequent vaccines We agree with this reviewer that we cannot rule out the possibility that these changes may have a negative impact for the production of some vaccines or other therapeutic products produced by M. pneumoniae. However, the changes observed are minor, suggesting at least the "potential" of this medium for production. In fact, experiments with this medium in the framework of the MycoSynVac project (https://www.mycosynvac.eu/), which aim to express specific recombinant proteins on the bacterial surface as a live vaccine, did not show a negative impact of expression (data not published). However, we agree that in other payloads it may have an impact. To note this, we have modified this sentence as follows in page 12, line 326-329: "Although quality assessment of the final bacterial product is required, these results show a minor impact in gene expression, supporting vB13 medium as a suitable and potential formulation for the production of M. pneumoniae-based therapies." (7) In this study, a serum-free medium was developed to replace the traditional medium. In the optimization process, whether there are still some natural ingredients in serum cannot be determined by artificial optimization?
The specific components in serum have not been fully identified, thus it is difficult to validate the specific contributions of natural ingredients of the serum. In addition, the concentration and combinatorial effects of these compounds are difficult to predict and assess by artificial optimization. The fact that our medium does not perform 100% as rich medium, suggests that there is still room for improvement, and we presume that transport proteins (such as lipoproteins) present in the serum are probably required for an efficient supply of lipids. In fact, lipid metabolism is by far the more limited biosynthetic pathway of M. pneumoniae, which entirely depends on external lipid sources. However, supplementation of these types of molecules would probably result in a medium containing components of animal origin, and increase considerably the cost of the medium in the case that these compounds would be commercially available. Therefore, although we agree that the identification of other natural ingredients needed from the serum is important, we believe that is difficult to achieve based on the metabolic network of M. pneumoniae, and probably incompatible with a medium suitable for industrial production.