The impact of sseK2 deletion on Salmonella enterica serovar typhimurium virulence in vivo and in vitro

Background Salmonella enterica is regarded as a major public health threat worldwide. Salmonella secretes the novel translocated effector protein K2 (SseK2), but it is unclear whether this protein plays a significant role in Salmonella enterica Typhimurium virulence. Results A ΔsseK2 mutant of S. Typhimurium exhibited similar growth curves, adhesion and invasive ability compared with wild-type (WT) bacteria. However, deletion of sseK2 rendered Salmonella deficient in biofilm formation and the early proliferative capacity of the ΔsseK2 mutant was significantly lower than that of the WT strain. In vivo, the LD50 (median lethal dose) of the ΔsseK2 mutant strain was increased 1.62 × 103-fold compared with the WT strain. In addition, vaccinating mice with the ΔsseK2 mutant protected them against challenge with a lethal dose of the WT strain. The ability of the ΔsseK2 mutant strain to induce systemic infection was highly attenuated compared with the WT strain, and the bacterial load in the animals’ internal organs was lower when they were infected with the ΔsseK2 mutant strain than when they were infected with the WT strain. Conclusions We conclude that sseK2 is a virulence-associated gene that plays a vital role in Salmonella virulence. Electronic supplementary material The online version of this article (10.1186/s12866-019-1543-2) contains supplementary material, which is available to authorized users.


INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.
Objectives 4 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

METHODS
Ethical statement 5 Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.

7
For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example: a. How (e.g. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). b. When (e.g. time of day). c. Where (e.g. home cage, laboratory, water maze). d. Why (e.g. rationale for choice of specific anaesthetic, route of administration, drug dose used).
Experimental animals 8 a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). b. Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g. knock-out or transgenic), genotype, health/immune status, drug or test naïve, previous procedures, etc. temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment). c. Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment.
Sample size 10 a. Specify the total number of animals used in each experiment, and the number of animals in each experimental group. b. Explain how the number of animals was arrived at. Provide details of any sample size calculation used. c. Indicate the number of independent replications of each experiment, if relevant.
Allocating animals to experimental groups 11 a. Give full details of how animals were allocated to experimental groups, including randomisation or matching if done. b. Describe the order in which the animals in the different experimental groups were treated and assessed.

12
Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).
Statistical methods 13 a. Provide details of the statistical methods used for each analysis. b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

Baseline data 14
For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).
Numbers analysed 15 a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50% 2 ).
b. If any animals or data were not included in the analysis, explain why.

Outcomes and estimation
16 Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval).
Adverse events 17 a. Give details of all important adverse events in each experimental group. b. Describe any modifications to the experimental protocols made to reduce adverse events. c. Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research.

Generalisability/ translation
19 Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology.
Funding 20 List all funding sources (including grant number) and the role of the funder(s) in the study.

Methods
Paragraph 1

Conclusion
Funding